Methods for improving yield using soybean event 3560.4.3.5

ABSTRACT

Compositions and methods related to transgenic glyphosate/ALS inhibitor-tolerant soybean plants are provided. Specifically, soybean plants having a 3560.4.3.5 event which imparts tolerance to glyphosate and at least one ALS-inhibiting herbicide are provided. The soybean plant harboring the 3560.4.3.5 event at the recited chromosomal location comprises genomic/transgene junctions having at least the polynucleotide sequence of SEQ ID NO:10 and/or 11. The characterization of the genomic insertion site of the 3560.4.3.5 event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the soybean 3560.4.3.5 events are provided. Methods and compositions for increasing yield are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation-in-Part of U.S. Utility applicationSer. No. 11/765,940, filed Jun. 20, 2007, which claims the benefit ofU.S. Provisional Application No. 60/817,011 filed on Jun. 28, 2006 andU.S. Provisional Application No. 60/847,154 filed on Sep. 26, 2006, eachwhich is herein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ON COMPACT DISK

The official copy of the sequence listing is submitted on compact disk(CD). Two CDs, labeled Copy 1 and Copy 2, containing an ASCII formattedsequence listing with a file name of 341203seqlist.txt, created on May30, 2008, and having a size of 88 KB, are filed concurrently with thespecification. The sequence listing contained on these compact disks ispart of the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to multiple herbicide tolerances conferred byexpression of a sequence that confers tolerance to glyphosate inconjunction with the expression of sequence that confers tolerance toone or more ALS inhibitor chemistries.

BACKGROUND OF THE INVENTION

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising et al.(1988) Ann. Rev. Genet. 22: 421-477). At the same time the presence ofthe transgene at different locations in the genome influences theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of events in order to identifyan event characterized by optimal expression of an introduced gene ofinterest. For example, it has been observed in plants and in otherorganisms that there may be a wide variation in levels of expression ofan introduced gene among events. There may also be differences inspatial or temporal patterns of expression, for example, differences inthe relative expression of a transgene in various plant tissues, thatmay not correspond to the patterns expected from transcriptionalregulatory elements present in the introduced gene construct. It is alsoobserved that the transgene insertion can affect the endogenous geneexpression. For these reasons, it is common to produce hundreds tothousands of different events and screen those events for a single eventthat has desired transgene expression levels and patterns for commercialpurposes. An event that has desired levels or patterns of transgeneexpression is useful for introgressing the transgene into other geneticbackgrounds by sexual outcrossing using conventional breeding methods.Progeny of such crosses maintain the transgene expressioncharacteristics of the original transformant. This strategy is used toensure reliable gene expression in a number of varieties that are welladapted to local growing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants, or for use in environmental monitoring,monitoring traits in crops in the field, or monitoring products derivedfrom a crop harvest, as well as, for use in ensuring compliance ofparties subject to regulatory or contractual terms.

In the commercial production of crops, it is desirable to easily andquickly eliminate unwanted plants (i.e., “weeds”) from a field of cropplants. An ideal treatment would be one which could be applied to anentire field but which would eliminate only the unwanted plants whileleaving the crop plants unharmed. One such treatment system wouldinvolve the use of crop plants which are tolerant to a herbicide so thatwhen the herbicide was sprayed on a field of herbicide-tolerant cropplants, the crop plants would continue to thrive whilenon-herbicide-tolerant weeds were killed or severely damaged. Ideally,such treatment systems would take advantage of varying herbicideproperties so that weed control could provide the best possiblecombination of flexibility and economy. For example, individualherbicides have different longevities in the field, and some herbicidespersist and are effective for a relatively long time after they areapplied to a field while other herbicides are quickly broken down intoother and/or non-active compounds. An ideal treatment system would allowthe use of different herbicides so that growers could tailor the choiceof herbicides for a particular situation.

Due to local and regional variation in dominant weed species as well aspreferred crop species, a continuing need exists for customized systemsof crop protection and weed management which can be adapted to the needsof a particular region, geography, and/or locality. Methods andcompositions that allow for the rapid identification of events in plantsthat produce such qualities are needed. For example, a continuing needexists for methods of crop protection and weed management which canreduce: the number of herbicide applications necessary to control weedsin a field; the amount of herbicide necessary to control weeds in afield; the amount of tilling necessary to produce a crop; and/orprograms which delay or prevent the development and/or appearance ofherbicide-resistant weeds. A continuing need exists for methods andcompositions of crop protection and weed management which allow thetargeted use of particular herbicide combinations and for the efficientdetection of such an event.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods related to transgenic glyphosate/ALSinhibitor-tolerant soybean plants are provided. Compositions comprisesoybean plants containing a 3560.4.3.5 event which imparts tolerance toglyphosate and at least one ALS-inhibiting herbicide. The soybean plantharboring the 3560.4.3.5 event at the recited chromosomal locationcomprises genomic/transgene junctions having at least the polynucleotidesequence of SEQ ID NO:10 and/or 11. Further provided are the seedsdeposited as Patent Deposit No. PTA-8287 and plants, plant cells, plantparts, grain and plant products derived therefrom. The characterizationof the genomic insertion site of event 3560.4.3.5 provides for anenhanced breeding efficiency and enables the use of molecular markers totrack the transgene insert in the breeding populations and progenythereof. Various methods and compositions for the identification,detection, and use of the soybean event 3560.4.3.5 are provided. Furtherprovided are methods and compositions that increase the yield of soybeanevent 3560.4.3.5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic map of PHP20163 indicating plasmid elementsand restriction enzyme sites for Not I, Asc I, Xba I and Hind III. Not Iand Asc I were used for isolation of fragment PHP20163A (FIG. 2).

FIG. 2 provides a schematic map of fragment PHP20163A indicatingrestriction enzyme sites for Xba I, Hind III and various geneticelements. This fragment was used for microprojectile bombardment toproduce the 3560.4.3.5 soybean event. Probes used for Southernhybridization are indicated as boxes beneath the fragment map and areidentified as follows: A: glyat4601 (glyphosate acetyltransferase) probeB: gm-hra probe (two non-overlapping segments were generated coveringthe entire region and used together for hybridization.

FIG. 3 provides a schematic map of the insertion region in soybean event3560.4.3.5 and shows the three regions that were sequenced from PCRproducts generated using genomic DNA as template: inserted DNA from thebombarded PHP20163A DNA fragment, 5′ flanking soybean genomic DNA and 3′flanking soybean genomic DNA.

FIG. 4A-E provides a complete sequence of DNA insert and flankinggenomic border regions in the 3560.4.3.5 soybean event (SEQ ID NO:6).The 5′ and 3′ flanking genomic border regions, bp 1 to 3317 and bp 8680to 10849, respectively, are underlined.

FIG. 5 provides a breeding diagram for 3560.4.3.5 soybean.

FIG. 6 provides a schematic map of fragment PHP20163A indicatinglocation of the genetic elements contained in the two gene expressioncassettes and base pair positions for Bgl II and Xba I restrictionenzyme sites. The Not I and Asc I restriction enzyme sites are lost uponexcision of this fragment from PHP20163. The total fragment size is 5362base pairs. Approximate locations of the probes used are shown asnumbered boxes below the fragment and are identified below. Additionaldetails on these probes are provided in Table 3.

FIG. 7 provides a schematic plasmid map of PHP20163 indicating thelocation of genetic elements and base pair positions for restrictionenzyme sites for Not I, Bgl II, Xba I, and Asc I. The Not I-Asc Ifragment of this plasmid was isolated (PHP20163A; map, FIG. 2) and usedfor transformation to produce 3560.4.3.5 soybean. The Xba I site locatedat bp 1387 contains a Dam methylase recognition site and is resistant todigestion by Xba I if the plasmid is prepared from a Dam⁺ strain. Thetotal plasmid size is 7954 base pairs. Backbone probes are indicatedschematically as lines within the plasmid diagram and are identifiedbelow. Each probe is comprised of two non-overlapping segments that werecombined for hybridization.

FIG. 8 provides a schematic map of the transgene insertion in 3560.4.3.5soybean based on Southern blot analysis. The flanking soybean genome isrepresented by the horizontal dotted line. A single, intact copy of thePHP20163A fragment integrated into the soybean genome. Bgl II and Xba Irestriction enzyme sites are indicated with the sizes of observedfragments on Southern blots shown below the map in base pairs (bp).

FIG. 9 provides a map of the 3560.4.3.5 event and depicts where variousprimers anneal.

FIG. 10A-I provides the left genomic border/internal transgeneinsert/right genomic border of the 3560.4.3.5 event (SEQ ID NO:6). Theregions where various primers anneal are illustrated.

FIG. 11 provides LSMean comparisons for yield (bu/ac) of ten differentpopulations of lines classified for glyphosate tolerance transgenes(GLYAT, EPSPS, GLYAT+EPSPS).

FIG. 12 provides LSMean comparisons for yield of two differentpopulations of related lines classified for glyphosate tolerancetransgenes (GLYAT, EPSPS, GLYAT+EPSPS).

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Compositions and methods related to transgenic glyphosate/ALSinhibitor-tolerant soybean plants are provided. Compositions includesoybean plants having event 3560.4.3.5. A soybean plant having “event3560.4.3.5” has been modified by the insertion of the glyphosateacetyltransferase (glyat) gene derived from Bacillus licheniformis and amodified version of the soybean acetolactate synthase gene (gm-hra). Theglyat gene was functionally improved by a gene shuffling process tooptimize the kinetics of glyphosate acetyltransferase (GLYAT) activityfor acetylating the herbicide glyphosate. The insertion of the glyatgene in the plant confers tolerance to the herbicidal active ingredientglyphosate through the conversion of glyphosate to the non-toxicacetylated form. The insertion of the gm-hra gene produces a modifiedform of the acetolactate synthase (ALS) enzyme. ALS is essential forbranched chain amino acid biosynthesis and is inhibited by certainherbicides. The modification in the gm-hra gene overcomes thisinhibition and thus provides tolerance to a wide range of ALS-inhibitingherbicides. Thus, a soybean plant having an event 3560.4.3.5 is tolerantto glyphosate and at least one ALS-inhibiting herbicide. The soybeanevent 3560.4.3.5 is otherwise known as Event DP-356043-5 or 356043soybean.

The polynucleotides conferring the glyphosate and ALS inhibitortolerance are linked on the same DNA construct and are inserted at acharacterized position in the soybean genome and thereby produce the3560.4.3.5 soybean event. The soybean plant harboring the 3560.4.3.5event at the recited chromosomal location comprises genomic/transgenejunctions having at least the polynucleotide sequence of SEQ ID NO:10and/or 11. The characterization of the genomic insertion site of the3560.4.3.5 event provides for an enhanced breeding efficiency andenables the use of molecular markers to track the transgene insert inthe breeding populations and progeny thereof. Various methods andcompositions for the identification, detection, and use of the soybean3560.4.3.5 events are provided herein. As used herein, the term “event3560.4.3.5 specific” refers to a polynucleotide sequence which issuitable for discriminatively identifying event 3560.4.3.5 in plants,plant material, or in products such as, but not limited to, food or feedproducts (fresh or processed) comprising, or derived from plantmaterial.

Compositions further include seed deposited as Patent Deposit Nos.PTA-8287 and plants, plant cells, and seed derived therefrom.Applicant(s) have made a deposit of at least 2500 seeds of soybean event3560.4.3.5 with the American Type Culture Collection (ATCC), Manassas,Va. 20110-2209 USA, on Mar. 27, 2007, and the deposits were assignedATCC Deposit No. PTA-8287. These deposits will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of patent Procedure. Thesedeposits were made merely as a convenience for those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The seeds deposited with the ATCC on Mar. 26, 2007 were taken fromthe deposit maintained by Pioneer Hi-Bred International, Inc., 7250 NW62^(nd) Avenue, Johnston, Iowa 50131-1000. Access to this deposit willbe available during the pendency of the application to the Commissionerof Patents and Trademarks and persons determined by the Commissioner tobe entitled thereto upon request. Upon allowance of any claims in theapplication, the Applicant(s) will make available to the public,pursuant to 37 C.F.R. §1.808, sample(s) of the deposit of at least 2500seeds of soybean event 3560.4.3.5 with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209.This deposit of seed of soybean event 3560.4.3.5 will be maintained inthe ATCC depository, which is a public depository, for a period of 30years, or 5 years after the most recent request, or for the enforceablelife of the patent, whichever is longer, and will be replaced if itbecomes nonviable during that period. Additionally, Applicant(s) havesatisfied all the requirements of 37 C.F.R. §§1.801-1.809, includingproviding an indication of the viability of the sample upon deposit.Applicant(s) have no authority to waive any restrictions imposed by lawon the transfer of biological material or its transportation incommerce. Applicant(s) do not waive any infringement of their rightsgranted under this patent or rights applicable to event 3560.4.3.5 underthe Plant Variety Protection Act (7 USC 2321 et seq.). Unauthorized seedmultiplication prohibited. The seed may be regulated.

As used herein, the term “soybean” means Glycine max and includes allplant varieties that can be bred with soybean. As used herein, the termplant includes plant cells, plant organs, plant protoplasts, plant celltissue cultures from which plants can be regenerated, plant calli, plantclumps, and plant cells that are intact in plants or parts of plantssuch as embryos, pollen, ovules, seeds, leaves, flowers, branches,fruit, stalks, roots, root tips, anthers, and the like. Grain isintended to mean the mature seed produced by commercial growers forpurposes other than growing or reproducing the species. Progeny,variants, and mutants of the regenerated plants are also included withinthe scope of the invention, provided that these parts comprise a3650.4.3.5 event.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s), including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. An eventis characterized phenotypically by the expression of the transgene(s).At the genetic level, an event is part of the genetic makeup of a plant.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety that include theheterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking DNA from the transformed parent ispresent in the progeny of the cross at the same chromosomal location.The term “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking sequence immediately adjacentto the inserted DNA that would be expected to be transferred to aprogeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can comprise either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500,or 5000 base pair or greater which is located either immediatelyupstream of and contiguous with or immediately downstream of andcontiguous with the original foreign insert DNA molecule. Non-limitingexamples of the flanking regions of the 3560.4.3.5 event are set forthin SEQ ID NO:4 and 5 and variants and fragments thereof.

Transformation procedures leading to random integration of the foreignDNA will result in transformants containing different flanking regionscharacteristic of and unique for each transformant. When recombinant DNAis introduced into a plant through traditional crossing, its flankingregions will generally not be changed. Transformants will also containunique junctions between a piece of heterologous insert DNA and genomicDNA, or two pieces of genomic DNA, or two pieces of heterologous DNA. A“junction” is a point where two specific DNA fragments join. Forexample, a junction exists where insert DNA joins flanking DNA. Ajunction point also exists in a transformed organism where two DNAfragments join together in a manner that is modified from that found inthe native organism. As used herein, “junction DNA” refers to DNA thatcomprises a junction point. Non-limiting examples of junction DNA fromthe 3560.4.3.5 event set are forth in SEQ ID NO:1, 2, 6, 10, 11, 12, 13,14, 15, 27, 28, 41, 42 or variants and fragments thereof.

A 3560.4.3.5 plant can be bred by first sexually crossing a firstparental soybean plant grown from the transgenic 3560.4.3.5 soybeanplant (or progeny thereof derived from transformation with theexpression cassettes of the embodiments that confer herbicide tolerance)and a second parental soybean plant that lacks the herbicide tolerancephenotype, thereby producing a plurality of first progeny plants; andthen selecting a first progeny plant that displays the desired herbicidetolerance; and selfing the first progeny plant, thereby producing aplurality of second progeny plants; and then selecting from the secondprogeny plants which display the desired herbicide tolerance. Thesesteps can further include the back-crossing of the first herbicidetolerant progeny plant or the second herbicide tolerant progeny plant tothe second parental soybean plant or a third parental soybean plant,thereby producing a soybean plant that displays the desired herbicidetolerance. It is further recognized that assaying progeny for phenotypeis not required. Various methods and compositions, as disclosedelsewhere herein, can be used to detect and/or identify the 3560.4.3.5event.

It is also to be understood that two different transgenic plants canalso be sexually crossed to produce offspring that contain twoindependently segregating added, exogenous genes. Selfing of appropriateprogeny can produce plants that are homozygous for both added, exogenousgenes. Back-crossing to a parental plant and out-crossing with anon-transgenic plant are also contemplated, as is vegetativepropagation. Descriptions of other breeding methods that are commonlyused for different traits and crops can be found in one of severalreferences, e.g., Fehr, in Breeding Methods for Cultivar Development,Wilcos J. ed., American Society of Agronomy, Madison Wis. (1987).

The term “germplasm” refers to an individual, a group of individuals, ora clone representing a genotype, variety, species or culture, or thegenetic material thereof.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generally isogenicor near isogenic.

Inbred soybean lines are typically developed for use in the productionof soybean hybrids and for use as germplasm in breeding populations forthe creation of new and distinct inbred soybean lines. Inbred soybeanlines are often used as targets for the introgression of novel traitsthrough traditional breeding and/or molecular introgression techniques.Inbred soybean lines need to be highly homogeneous, homozygous andreproducible to be useful as parents of commercial hybrids. Manyanalytical methods are available to determine the homozygosity andphenotypic stability of inbred lines.

The phrase “hybrid plants” refers to plants which result from a crossbetween genetically different individuals.

The term “crossed” or “cross” in the context of this invention means thefusion of gametes, e.g., via pollination to produce progeny (i.e.,cells, seeds, or plants) in the case of plants. The term encompassesboth sexual crosses (the pollination of one plant by another) and, inthe case of plants, selfing (self-pollination, i.e., when the pollenand, ovule are from the same plant).

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. In onemethod, the desired alleles can be introgressed through a sexual crossbetween two parents, wherein at least one of one of the parents has thedesired allele in its genome.

In some embodiments, the polynucleotide conferring the soybean3560.4.3.5 event of the invention are engineered into a molecular stack.In other embodiments, the molecular stack further comprises at least oneadditional polynucleotide that confers tolerance to a 3^(rd) herbicide.In one embodiment, the sequence confers tolerance to glufosinate, and ina specific embodiment, the sequence comprises pat.

In other embodiments, the soybean 3560.4.3.5 event of the inventioncomprise one or more trait of interest, and in more specificembodiments, the plant is stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredcombination of traits. A trait, as used herein, refers to the phenotypederived from a particular sequence or groups of sequences. For example,herbicide-tolerance polynucleotides may be stacked with any otherpolynucleotides encoding polypeptides having pesticidal and/orinsecticidal activity, such as Bacillus thuringiensis toxic proteins(described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; Geiser et al. (1986) Gene 48: 109; Lee et al. (2003) Appl.Environ. Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al. (2001) ActaCrystallogr. D. Biol. Crystallogr. 57: 1101-1109 (Cry3Bb1); and Hermanet al. (2004) J. Agric. Food Chem. 52: 2726-2734 (Cry1F)), lectins (VanDamme et al. (994) Plant Mol. Biol. 24: 825, pentin (described in U.S.Pat. No. 5,981,722), and the like. The combinations generated can alsoinclude multiple copies of any one of the polynucleotides of interest.

In some embodiments, soybean 3560.4.3.5 event may further comprise otherherbicide-tolerance traits to create a transgenic plant of the inventionwith further improved properties. In specific embodiments, theadditional herbicide-tolerance traits are stacked with the 3560.4.3.5event. Other herbicide-tolerance polynucleotides that could be used insuch embodiments include those conferring tolerance to glyphosate or toALS inhibitors by other modes, of action, such as, for example, a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175. Other traits that could becombined with the soybean 3560.4.3.5 events include those derived frompolynucleotides that confer on the plant the capacity to produce ahigher level of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), forexample, as more fully described in U.S. Pat. Nos. 6,248,876 B1;5,627,061; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910;5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and5,491,288; and international publications WO 97/04103; WO 00/66746; WO01/66704; and WO 00/66747. Other traits that could be combined with thesoybean 3560.4.3.5 event include those conferring tolerance tosulfonylurea and/or imidazolinone, for example, as described more fullyin U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180;5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; andinternational publication WO 96/33270.

Additional EPSPS sequences that are tolerant to glyphosate are describedin U.S. Pat. Nos. 6,248,876; 5,627,061; 5,804,425; 5,633,435; 5,145,783;4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114;6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re.36,449; RE 37,287 E; and 5,491,288; and international publications WO97/04103; WO 00/66746; WO 01/66704; and WO 00/66747; 6,040,497;5,094,945; 5,554,798; 6,040,497; Zhou et al. (1995) Plant CellRep.:159-163; WO 0234946; WO 9204449; 6,225,112; 4,535,060, and6,040,497, which are incorporated herein by reference in theirentireties for all purposes. Additional EPSP synthase sequences include,gdc-1 (U.S. App. Publication 20040205847); EPSP synthases with class IIIdomains (U.S. App. Publication 20060253921); gdc-1 (U.S. App.Publication 20060021093); gdc-2 (U.S. App. Publication 20060021094);gro-1 (U.S. App. Publication 20060150269); grg23 or grg 51 (U.S. App.Publication 20070136840); GRG32 (U.S. App. Publication 20070300325);GRG33, GRG35, GRG36, GRG37, GRG38, GRG39 and GRG50 (U.S. App.Publication 20070300326); or EPSP synthase sequences disclosed in, U.S.App. Publication 20040177399; 20050204436; 20060150270; 20070004907;20070044175; 2007010707; 20070169218; 20070289035; and, 20070295251;each of which is herein incorporated by reference in their entirety.

In one non-limiting embodiment, the glyphosate-tolerant EPSPS sequenceemployed is the EPSPS polypeptide from Agrobacterium sp. Strain CP4 asdescribed in Pagette et al (1995) Development, Identification, andCharacterization of a Glyphosate-Tolerance Soybean Line. Crop Sci.35:1451-1461, herein incorporated by reference in its entirety. See,also GenBank Accession number Q9R4E4, herein incorporated by referenceand set forth in SEQ ID NO: 56. In still further embodiments, the EPSPSsequence from the glyphosate tolerant soybean line 40-3-2 is combinedwith a GLYAT sequence in planta. Other EPSPS events of interest includeMON-89788-1 (MON89788). See, www.agbios.com/main.php.

In some embodiments, the soybean 3560.4.3.5 event may be combined orstacked with, for example, hydroxyphenylpyruvatedioxygenases which areenzymes that catalyze the reaction in which para-hydroxyphenylpyruvate(HPP) is transformed into homogentisate. Molecules which inhibit thisenzyme and which bind to the enzyme in order to inhibit transformationof the HPP into homogentisate are useful as herbicides. Traitsconferring tolerance to such herbicides in plants are described in U.S.Pat. Nos. 6,245,968 B1; 6,268,549; and 6,069,115; and internationalpublication WO 99/23886. Other examples of suitable herbicide-tolerancetraits that could be stacked with the soybean 3560.4.3.5 event includearyloxyalkanoate dioxygenase polynucleotides (which reportedly confertolerance to 2,4-D and other phenoxy auxin herbicides as well as toaryloxyphenoxypropionate herbicides as described, for example, inWO2005/107437) and dicamba-tolerance polynucleotides as described, forexample, in Herman et al. (2005) J. Biol. Chem. 280: 24759-24767.

Other examples of herbicide-tolerance traits that could be combined orstacked with the soybean 3560.4.3.5 event include those conferred bypolynucleotides encoding an exogenous phosphinothricinacetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616; and 5,879,903. Plants containing an exogenousphosphinothricin acetyltransferase can exhibit improved tolerance toglufosinate herbicides, which inhibit the enzyme glutamine synthase.Other examples of herbicide-tolerance traits that could be combined withthe soybean 3560.4.3.5 event include those conferred by polynucleotidesconferring altered protoporphyrinogen oxidase (protox) activity, asdescribed in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373;and international publication WO 01/12825. Plants containing suchpolynucleotides can exhibit improved tolerance to any of a variety ofherbicides which target the protox enzyme (also referred to as “protoxinhibitors”).

Other examples of herbicide-tolerance traits that could be combined orstacked with the soybean 3560.4.3.5 event include those conferringtolerance to at least one herbicide in a plant such as, for example, asoybean plant or horseweed. Herbicide-tolerant weeds are known in theart, as are plants that vary in their tolerance to particularherbicides. See, e.g., Green and Williams (2004) “Correlation of Corn(Zea mays) Inbred Response to Nicosulfuron and Mesotrione,” posterpresented at the WSSA Annual Meeting in Kansas City, Mo., Feb. 9-12,2004; Green (1998) Weed Technology 12: 474-477; Green and Ulrich (1993)Weed Science 41: 508-516. The trait(s) responsible for these tolerancescan be combined by breeding or via other methods with the soybean3560.4.3.5 event to provide a plant of the invention as well as methodsof use thereof.

The soybean 3560.4.3.5 event can also be combined or stacked with atleast one other trait to produce plants of the present invention thatfurther comprise a variety of desired trait combinations including, butnot limited to, traits desirable for animal feed such as high oilcontent (e.g., U.S. Pat. No. 6,232,529); balanced amino acid content(e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802;and 5,703,409; U.S. Pat. No. 5,850,016); barley high lysine (Williamsonet al. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279;Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) PlantMol. Biol. 12:123)); increased digestibility (e.g., modified storageproteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); andthioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001)); the disclosures of which are herein incorporated by reference.Desired trait combinations also include LLNC (low linolenic acidcontent; see, e.g., Dyer et al. (2002) Appl. Microbiol. Biotechnol. 59:224-230) and OLCH (high oleic acid content; see, e.g., Fernandez-Moya etal. (2005) J. Agric. Food Chem. 53: 5326-5330).

The soybean 3560.4.3.5 event can also be combined or stacked with otherdesirable traits such as, for example, fumonisim detoxification genes(U.S. Pat. No. 5,792,931), avirulence and disease resistance genes(Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable forprocessing or process products such as modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE));and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference. One could also combineherbicide-tolerant polynucleotides with polynucleotides providingagronomic traits such as male sterility (e.g., see U.S. Pat. No.5,583,210), stalk strength, flowering time; or transformation technologytraits such as cell cycle regulation or gene targeting (e.g., WO99/61619, WO 00/17364, and WO 99/25821); the disclosures of which areherein incorporated by reference.

In another embodiment, the soybean 3560.4.3.5 event can also be combinedor stacked with the Rcg1 sequence or biologically active variant orfragment thereof. The Rcg1 sequence is an anthracnose stalk rotresistance gene in corn. See, for example, U.S. patent application Ser.No. 11/397,153, 11/397,275, and 11/397,247, each of which is hereinincorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

As used herein, the use of the term “polynucleotide” is not intended tolimit a polynucleotide to comprise DNA. Those of ordinary skill in theart will recognize that polynucleotides, can comprise ribonucleotidesand combinations of ribonucleotides and deoxyribonucleotides. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The polynucleotides alsoencompass all forms of sequences including, but not limited to,single-stranded forms, double-stranded forms, hairpins, stem-and-loopstructures, and the like.

A 3560.4.3.5 plant comprises an expression cassette having a glyphosateacetyltransferase polynucleotide and a genetically modified acetolactatesynthase polynucleotide (gm-hra). The cassette can include 5′ and 3′regulatory sequences operably linked to the glyat and the gm-hrapolynucleotides. “Operably linked” is intended to mean a functionallinkage between two or more elements. For example, an operable linkagebetween a polynucleotide of interest and a regulatory sequence (i.e., apromoter) is functional link that allows for the expression of thepolynucleotide of interest. Operably linked elements may be contiguousor non-contiguous. When used to refer to the joining of two proteincoding regions, by operably linked it is intended that the codingregions are in the same reading frame. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes. Such an expression cassette is providedwith a plurality of restriction sites and/or recombination sites forinsertion of the polynucleotide to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a coding region, and a transcriptional andtranslational termination region functional in plants. “Promoter” refersto a nucleotide sequence capable of controlling the expression of acoding sequence or functional RNA. In general, a coding sequence islocated 3′ to a promoter sequence. The promoter sequence can compriseproximal and more distal upstream elements, the latter elements areoften referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The expression cassettes may also contain 5′ leader sequences. Suchleader sequences can act to enhance translation. The regulatory regions(i.e., promoters, transcriptional regulatory regions, RNA processing orstability regions, introns, polyadenylation signals, and translationaltermination regions) and/or the coding region may be native/analogous orheterologous to the host cell or to each other.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency. Examples of translation leader sequences have been described(Turner and Foster (1995) Mol. Biotechnol. 3: 225-236). The “3′non-coding sequences” refer to nucleotide sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1: 671-680.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved. The expression cassette can alsocomprise a selectable marker gene for the selection of transformedcells. Selectable marker genes are utilized for the selection oftransformed cells or tissues.

Isolated polynucleotides are provided that can be used in variousmethods for the detection and/or identification of the soybean3560.4.3.5 event. An “isolated” or “purified” polynucleotide, orbiologically active portion thereof, is substantially or essentiallyfree from components that normally accompany or interact with thepolynucleotide as found in its naturally occurring environment. Thus, anisolated or purified polynucleotide is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived.

In specific embodiments, the polynucleotides comprise the junction DNAsequence set forth in SEQ ID NO:10 or 11. In other embodiments, thepolynucleotides comprise the junction DNA sequences set forth in SEQ IDNO:1, 2, 6, 12, 13, 14, 15, 27, 28, 41 or 42 or variants and fragmentsthereof. Fragments and variants of junction DNA sequences are suitablefor discriminatively identifying event 3560.4.3.5. As discussedelsewhere herein, such sequences find use as primers and/or probes.

In other embodiments, the polynucleotides are provided that can detect a3560.4.3.5 event or a 3560.4.3.5 specific region. Such sequences includeany polynucleotide set forth in SEQ ID NOS:1-56 or variants andfragments thereof. In specific embodiments, the polynucleotide used todetect a 3560.4.3.5 event comprise the sequence set forth in SEQ ID NO:43 or a fragment of SEQ ID NO:43 having at least 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 nucleotides.Fragments and variants of polynucleotides that detect a 3560.4.3.5 eventor a 3560.4.3.5 specific region are suitable for discriminativelyidentifying event 3560.4.3.5. As discussed elsewhere herein, suchsequences find use as primers and/or probes. Further provided areisolated DNA nucleotide primer sequences comprising or consisting of asequence set forth in SEQ ID NO:7, 8, 9, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 37, 38, 39, 40, 43, 44, 45, 46, 51, 52, 53, 54, or 55 or acomplement thereof or variants and fragments of SEQ ID NO:1, 2, 3, 4, 5,6, 10, 11, 12, 13, 14, 15, 27, 28, 41, 42, or 43.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having deletions(i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition ofone or more nucleotides at one or more internal sites in the nativepolynucleotide; and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide.

As used herein, a “probe” is an isolated polynucleotide to which isattached a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, enzyme, etc. Such aprobe is complementary to a strand of a target polynucleotide, in theinstant case, to a strand of isolated DNA from soybean event 3560.4.3.5whether from a soybean plant or from a sample that includes DNA from theevent. Probes include not only deoxyribonucleic or ribonucleic acids butalso polyamides and other probe materials that can specifically detectthe presence of the target DNA sequence.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs refer to their use for amplification of atarget polynucleotide, e.g., by the polymerase chain reaction (PCR) orother conventional nucleic-acid amplification methods. “PCR” or“polymerase chain reaction” is a technique used for the amplification ofspecific DNA segments (see, U.S. Pat. Nos. 4,683,195 and 4,800,159;herein incorporated by reference). Any combination of primers disclosedherein can be used such that the pair allows for the detection a3560.4.3.5 event or specific region (i.e., SEQ ID NOS: 7-9, 16-26, 37,38, 39, 40, 44-46, or 51-55). Non-limiting examples of primer pairsinclude SEQ ID NOS:16 and 17; SEQ ID NOS:23 and 20; SEQ ID NOS:23 and19; SEQ ID NOS:18 AND 22; SEQ ID NOS:21 and 22; SEQ ID NO: 7 and 9; SEQID NO:8 and 9; SEQ ID NO:7 and 8; SEQ ID NO:37 and 39; and SEQ ID NO:38and 39; and SEQ ID NO: 44 and 45 and SEQ ID NOS: 37 and 38.

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide having a 3560.4.3.5 event. It is recognized that thehybridization conditions or reaction conditions can be determined by theoperator to achieve this result. This length may be of any length thatis of sufficient length to be useful in a detection method of choice.Generally, 8, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 75, 100,200, 300, 400, 500, 600, 700 nucleotides or more, or between about11-20, 20-30, 30-40, 40-50, 50-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800, or more nucleotides in length are used. Suchprobes and primers can hybridize specifically to a target sequence underhigh stringency hybridization conditions. Probes and primers accordingto embodiments may have complete DNA sequence identity of contiguousnucleotides with the target sequence, although probes differing from thetarget DNA sequence and that retain the ability to specifically detectand/or identify a target DNA sequence may be designed by conventionalmethods. Accordingly, probes and primers can share about 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identityor complementarity to the target polynucleotide (i.e., SEQ ID NO:1-46and 47-55), or can differ from the target sequence (i.e., SEQ ID NO:1-46 and 47-55) by 1, 2, 3, 4, 5, 6 or more nucleotides. Probes can beused as primers, but are generally designed to bind to the target DNA orRNA and are not used in an amplification process.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” or can itselfbe detected for identifying event 3560.4.3.5 in biological samples.Alternatively, a probe can be used during the PCR reaction to allow forthe detection of the amplification event (i.e., a Taqman probe or a MGBprobe) (so called real time PCR). When the probe is hybridized with thepolynucleotides of a biological sample under conditions which allow forthe binding of the probe to the sample, this binding can be detected andthus allow for an indication of the presence of event 3560.4.3.5 in thebiological sample. Such identification of a bound probe has beendescribed in the art. In an embodiment, the specific probe is a sequencewhich, under optimized conditions, hybridizes specifically to a regionwithin the 5′ or 3′ flanking region of the event and also comprises apart of the foreign DNA contiguous therewith. The specific probe maycomprise a sequence of at least 80%, between 80 and 85%, between 85 and90%, between 90 and 95%, and between 95 and 100% identical (orcomplementary) to a specific region of the 3560.4.3.5 event.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleotide amplification of a target polynucleotide that is part ofa nucleic acid template. For example, to determine whether a soybeanplant resulting from a sexual cross contains the 3560.4.3.5 event, DNAextracted from the soybean plant tissue sample may be subjected to apolynucleotide amplification method using a DNA primer pair thatincludes a first primer derived from flanking sequence adjacent to theinsertion site of inserted heterologous DNA, and a second primer derivedfrom the inserted heterologous DNA to produce an amplicon that isdiagnostic for the presence of the 3560.4.3.5 event DNA. By “diagnostic”for a 3650.4.3.5 event the use of any method or assay whichdiscriminates between the presence or the absence of a 3560.4.3.5 eventin a biological sample is intended. Alternatively, the second primer maybe derived from the flanking sequence. In still other embodiments,primer pairs can be derived from flanking sequence on both sides of theinserted DNA so as to produce an amplicon that includes the entireinsert polynucleotide of the expression construct as well as thesequence flanking the transgenic insert. See, FIG. 1. The amplicon is ofa length and has a sequence that is also diagnostic for the event (i.e.,has a junction DNA from a 3560.4.3.5 event). The amplicon may range inlength from the combined length of the primer pairs plus one nucleotidebase pair to any length of amplicon producible by a DNA amplificationprotocol. A member of a primer pair derived from the flanking sequencemay be located a distance from the inserted DNA sequence, this distancecan range from one nucleotide base pair up to the limits of theamplification reaction, or about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol.1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as the PCR primeranalysis tool in Vector NTI version 6 (Informax Inc., Bethesda Md.);PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer (Version0.5.COPYRGT., 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.). Additionally, the sequence can be visually scannedand primers manually identified using guidelines known to one of skillin the art.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant, thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143: 277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327: 70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below.

Thus, isolated polynucleotides can be incorporated into recombinantconstructs, typically DNA constructs, which are capable of introductioninto and replication in a host cell. Such a construct can be a vectorthat includes a replication system and sequences that are capable oftranscription and translation of a polypeptide-encoding sequence in agiven host cell. A number of vectors suitable for stable transfection ofplant cells or for the establishment of transgenic plants have beendescribed in, e.g., Pouwels et al. (1985; Supp. 1987) Cloning Vectors: ALaboratory Manual, Weissbach and Weissbach (1989) Methods for PlantMolecular Biology (Academic Press, New York); and Flevin et al. (1990)Plant Molecular Biology Manual (Kluwer Academic Publishers). Typically,plant expression vectors include, for example, one or more cloned plantgenes under the transcriptional control of 5′ and 3′ regulatorysequences and a dominant selectable marker. Such plant expressionvectors also can contain a promoter regulatory region (e.g., aregulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Various methods and compositions for identifying event 3560.4.3.5 areprovided. Such methods find use in identifying and/or detecting a3560.4.3.5 event in any biological material. Such methods include, forexample, methods to confirm seed purity and methods for screening seedsin a seed lot for a 3560.4.3.5 event. In one embodiment, a method foridentifying event 3560.4.3.5 in a biological sample is provided andcomprises contacting the sample with a first and a second primer; and,amplifying a polynucleotide comprising a 3560.4.3.5 specific region.

A biological sample can comprise any sample in which one desires todetermine if DNA having event 3560.4.3.5 is present. For example, abiological sample can comprise any plant material or material comprisingor derived from a plant material such as, but not limited to, food orfeed products. As used herein, “plant material” refers to material whichis obtained or derived from a plant or plant part. In specificembodiments, the biological sample comprises a soybean tissue.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences. The polynucleotide probes and primersspecifically detect a target DNA sequence. Any conventional nucleic acidhybridization or amplification method can be used to identify thepresence of DNA from a transgenic event in a sample. By “specificallydetect” it is intended that the polynucleotide can be used either as aprimer to amplify a 3560.4.3.5 specific region or the polynucleotide canbe used as a probe that hybridizes under stringent conditions to apolynucleotide having a 3560.4.3.5 event or a 3560.4.3.5 specificregion. The level or degree of hybridization which allows for thespecific detection of a 3560.4.3.5 event or a specific region of a3560.4.3.5 event is sufficient to distinguish the polynucleotide withthe 3560.4.3.5 specific region from a polynucleotide lacking this regionand thereby allow for discriminately identifying a 3560.4.3.5 event. By“shares sufficient sequence identity or complementarity to allow for theamplification of a 3560.4.3.5 specific event” is intended the sequenceshares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity or complementarity to a fragment or across the fulllength of the polynucleotide having the 3560.4.3.5 specific region.

Regarding the amplification of a target polynucleotide (e.g., by PCR)using a particular amplification primer pair, “stringent conditions” areconditions that permit the primer pair to hybridize to the targetpolynucleotide to which a primer having the corresponding wild-typesequence (or its complement) would bind and preferably to produce anidentifiable amplification product (the amplicon) having a 3560.4.3.5specific region in a DNA thermal amplification reaction. In a PCRapproach, oligonucleotide primers can be designed for use in PCRreactions to amplify a 3560.4.3.5 specific region. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Methods ofamplification are further described in U.S. Pat. Nos. 4,683,195,4,683,202 and Chen et al. (1994) PNAS 91:5695-5699. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the other embodiments. It is understood that a numberof parameters in a specific PCR protocol may need to be adjusted tospecific laboratory conditions and may be slightly modified and yetallow for the collection of similar results. These adjustments will beapparent to a person skilled in the art.

The amplified polynucleotide (amplicon) can be of any length that allowsfor the detection of the 3560.4.3.5 event or a 3560.4.3.5 specificregion. For example, the amplicon can be about 10, 50, 100, 200, 300,500, 700, 100, 2000, 3000, 4000, 5000 nucleotides in length or longer.

In specific embodiments, the specific region of the 3560.4.3.5 event isdetected.

Any primer that allows a 3560.4.3.5 specific region to be amplifiedand/or detected can be employed in the methods. For example, in specificembodiments, the first primer comprises a fragment of a polynucleotideof SEQ ID NO: 4 or 5, wherein the first or the second primer sharessufficient sequence identity or complementarity to the polynucleotide toamplify the 3560.4.3.5 specific region. The primer pair can comprise afragment of SEQ ID NO:4 and a fragment of SEQ ID NO:5 or 3, oralternatively, the primer pair can comprise a fragment of SEQ ID NO:5and a fragment of SEQ ID NO: 3 or 4. In still further embodiments, thefirst and the second primer can comprise any one or any combination ofthe sequences set forth in SEQ ID NO:7, 8, 9, 16-26, 37, 38, 39, 40,44-46, or 51-55. The primers can be of any length sufficient to amplifya 3560.4.3.5 region including, for example, at least 6, 7, 8, 9, 10, 15,20, 15, or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40,40-45 nucleotides or longer.

As discussed elsewhere herein, any method to PCR amplify the 3560.4.3.5event or specific region can be employed, including for example, realtime PCR. See, for example, Livak et al. (1995) PCR Methods andApplications 4:357-362; U.S. Pat. No. 5,538,848; U.S. Pat. No.5,723,591; Applied Biosystems User Bulletin No. 2, “RelativeQuantitation of Gene Expression,” P/N 4303859; and, Applied BiosystemsUser Bulletin No. 5, “Multiplex PCR with Taqman VIC probes,” P/N4306236; each of which is herein incorporated by reference.

Thus, in specific embodiments, a method of detecting the presence ofsoybean event 3560.4.3.5 or progeny thereof in a biological sample isprovided. The method comprises (a) extracting a DNA sample from thebiological sample; (b) providing a pair of DNA primer molecules (i.e,any combination of SEQ ID NOS: 7-9, 16-26, 37, 38, 39, 40, 44-46, or51-55, wherein said combination amplifies a 3560.4.3.5 event),including, but not limited to, i) the sequences of SEQ ID NO:16 and SEQID NO:17, ii) the sequences of SEQ ID NO:23 and SEQ ID NO:20; iii) thesequences of SEQ ID NO:23 and SEQ ID NO:19; iv) the sequences of SEQ IDNO:18 and SEQ ID NO:22; v) SEQ ID NO:21 and SEQ ID NO:22; vi) SEQ ID NO:7 and 9; vii) SEQ ID NO: 8 and 9; iix) SEQ ID NO: 7 and 8; ix) SEQ IDNO: 37 and 39; x) SEQ ID NO: 38 and 39; xi) SEQ ID NO: 44 and 45; xii)SEQ ID NO: 25 and 26; xiii) SEQ ID NO:25 and SEQ ID NO:24; (c) providingDNA amplification reaction conditions; (d) performing the DNAamplification reaction, thereby producing a DNA amplicon molecule; and(e) detecting the DNA amplicon molecule, wherein the detection of saidDNA amplicon molecule in the DNA amplification reaction indicates thepresence of soybean event 3560.4.3.5. In order for a nucleic acidmolecule to serve as a primer or probe it needs only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

In hybridization techniques, all or part of a polynucleotide thatselectively hybridizes to a target polynucleotide having a 3560.4.3.5specific event is employed. By “stringent conditions” or “stringenthybridization conditions” when referring to a polynucleotide probeconditions under which a probe will hybridize to its target sequence toa detectably greater degree than to other sequences (e.g., at least2-fold over background) are intended. Regarding the amplification of atarget polynucleotide (e.g., by PCR) using a particular amplificationprimer pair, “stringent conditions” are conditions that permit theprimer pair to hybridize to the target polynucleotide to which a primerhaving the corresponding wild-type. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of identity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is apolynucleotide that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions for hybridization and detection will bethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl (1984)Anal. Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61(% form)−500/L; where M is the molarity of monovalent cations, % GC isthe percentage of guanosine and cytosine nucleotides in the DNA, % formis the percentage of formamide in the hybridization solution, and L isthe length of the hybrid in base pairs. The T_(m) is the temperature(under defined ionic strength and pH) at which 50% of a complementarytarget sequence hybridizes to a perfectly matched probe. T_(m) isreduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with ≧90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Haymes et al. (1985) In: NucleicAcid Hybridization, a Practical Approach, IRL Press, Washington, D.C.

A polynucleotide is said to be the “complement” of anotherpolynucleotide if they exhibit complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the polynucleotide molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions.

Further provided are methods of detecting the presence of DNAcorresponding to the 3560.4.3.5 event in a sample. In one embodiment,the method comprises (a) contacting the biological sample with apolynucleotide probe that hybridizes under stringent hybridizationconditions with DNA from soybean event 3560.4.3.5 and specificallydetects the 3560.4.3.5 event; (b) subjecting the sample and probe tostringent hybridization conditions; and (c) detecting hybridization ofthe probe to the DNA, wherein detection of hybridization indicates thepresence of the 3560.4.3.5 event. In one embodiment, the DNA is digestedwith appropriate enzymes are preformed prior to the hybridization event.

Various method can be used to detect the 3560.4.3.5 specific region oramplicon thereof, including, but not limited to, Genetic Bit Analysis(Nikiforov et al. (1994) Nucleic Acid Res. 22: 4167-4175). In onemethod, a DNA oligonucleotide is designed which overlaps both theadjacent flanking DNA sequence and the inserted DNA sequence. In otherembodiments, DNA oligos are designed to allow for a 3560.4.3.5 specificamplicon. The oligonucleotide is immobilized in wells of a microwellplate. Following PCR of the region of interest a single-stranded PCRproduct can be hybridized to the immobilized oligonucleotide and serveas a template for a single base extension reaction using a DNApolymerase and labeled ddNTPs specific for the expected next base.Readout may be fluorescent or ELISA-based. A signal indicates presenceof the insert/flanking sequence due to successful amplification,hybridization, and single base extension.

Another detection method is the Pyrosequencing technique as described byWinge ((2000) Innov. Pharma. Tech. 00: 18-24). In this method, anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction or a pair of oligos are employed that can amplify a3560.4.3.5 specific region. The oligonucleotide is hybridized to asingle-stranded PCR product from the region of interest (one primer inthe inserted sequence and one in the flanking sequence) and incubated inthe presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. dNTPs are added individuallyand the incorporation results in a light signal which is measured. Alight signal indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single ormulti-base extension.

Fluorescence Polarization as described by Chen et al. ((1999) GenomeRes. 9: 492-498, 1999) is also a method that can be used to detect anamplicon of the invention. Using this method, an oligonucleotide isdesigned which overlaps the flanking and inserted DNA junction or a pairof oligos are employed that can amplify a 3560.4.3.5 specific region.The oligonucleotide is hybridized to a single-stranded PCR product fromthe region of interest (one primer in the inserted DNA and one in theflanking DNA sequence) and incubated in the presence of a DNA polymeraseand a fluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction or a pair of oligos are employed thatcan amplify a 3560.4.3.5 specific region. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. ((1996) Nature Biotech. 14: 303-308).Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking and insert DNA junction or a pair of oligos are employed thatcan amplify a 3560.4.3.5 specific region. The unique structure of theFRET probe results in it containing secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

As used herein, “kit” refers to a set of reagents for the purpose ofperforming the method embodiments, more particularly, the identificationand/or the detection of the 3560.4.3.5 event in biological samples. Thekit can be used, and its components can be specifically adjusted, forpurposes of quality control (e.g. purity of seed lots), detection ofevent 3560.4.3.5 in plant material, or material comprising or derivedfrom plant material, such as but not limited to food or feed products.

In specific embodiments, a kit for identifying event 3560.4.3.5 in abiological sample is provided. The kit comprises a first and a secondprimer, wherein the first and second primer amplify a polynucleotidecomprising a 3560.4.3.5 specific region. In further embodiments, the kitalso comprises a polynucleotide for the detection of the 3560.4.3.5specific region. The kit can comprise, for example, a first primercomprising a fragment of a polynucleotide of SEQ ID NO:4 or 5, whereinthe first or the second primer shares sufficient sequence homology orcomplementarity to the polynucleotide to amplify said 3560.4.3.5specific region. For example, in specific embodiments, the first primercomprises a fragment of a polynucleotide of SEQ ID NO:4 or 5, whereinthe first or the second primer shares sufficient sequence homology orcomplementarity to the polynucleotide to amplify said 3560.4.3.5specific region. The primer pair can comprises a fragment of SEQ ID NO:4and a fragment of SEQ ID NO:5 or 3, or alternatively, the primer paircan comprises a fragment of SEQ ID NO:5 and a fragment of SEQ ID NO:3 or4. In still further embodiments, the first and the second primer cancomprise any one or any combination of the sequences set forth in SEQ IDNO:7, 8, 9, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 37, 38, 39, 40,43, 44, 45, 46, or 51-55. The primers can be of any length sufficient toamplify the 3560.4.3.5 region including, for example, at least 6, 7, 8,9, 10, 15, 20, 15, or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30,30-35, 35-40, 40-45 nucleotides or longer.

Further provided are DNA detection kits comprising at least onepolynucleotide that can specifically detect a 3560.4.3.5 specificregion, wherein said polynucleotide comprises at least one DNA moleculeof a sufficient length of contiguous nucleotides homologous orcomplementary to SEQ ID NO: 3, 4 or 5. In specific embodiments, the DNAdetection kit comprises a polynucleotide having SEQ ID NO:10 or 11 orcomprises a sequence which hybridizes with sequences selected from thegroup consisting of: a) the sequences of SEQ ID NO: 4 and SEQ ID NO:3;and, b) the sequences of SEQ ID NO: 5 and SEQ ID NO: 3, and a sequenceof SEQ ID NO:4 and 43.

Any of the polynucleotides and fragments and variants thereof employedin the methods and compositions can share sequence identity to a regionof the transgene insert of the 3560.4.3.5 event, a junction sequence ofthe 3560.4.3.5 event or a flanking sequence of the 3560.4.3.5 event.Methods to determine the relationship of various sequences are known. Asused herein, “reference sequence” is a defined sequence used as a basisfor sequence comparison. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two polynucleotides. Generally, the comparison window is at least20 contiguous nucleotides in length, and optionally can be 30, 40, 50,100, or longer. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to a protein orpolypeptide. To obtain gapped alignments for comparison purposes, GappedBLAST (in BLAST 2.0) can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST2.0) can be used to perform an iterated search that detects distantrelationships between molecules. See Altschul et al. (1997) supra. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10 is intended.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin. GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the Quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Methods are provided for controlling weeds in an area of cultivation,preventing the development or the appearance of herbicide resistantweeds in an area of cultivation, producing a crop, and increasing cropsafety. The term “controlling,” and derivations thereof, for example, asin “controlling weeds” refers to one or more of inhibiting the growth,germination, reproduction, and/or proliferation of; and/or killing,removing, destroying, or otherwise diminishing the occurrence and/oractivity of a weed.

As used herein, an “area of cultivation” comprises any region in whichone desires to grow a plant. Such areas of cultivations include, but arenot limited to, a field in which a plant is cultivated (such as a cropfield, a sod field, a tree field, a managed forest, a field forculturing fruits and vegetables, etc), a greenhouse, a growth chamber,etc.

The methods comprise planting the area of cultivation with the soybean3560.4.3.5 seeds or plants, and in specific embodiments, applying to thecrop, seed, weed or area of cultivation thereof an effective amount of aherbicide of interest. It is recognized that the herbicide can beapplied before or after the crop is planted in the area of cultivation.Such herbicide applications can include an application of glyphosate, anALS inhibitor chemistry, or any combination thereof. In specificembodiments, a mixture of an ALS inhibitor chemistry in combination withglyphosate is applied to the soybean 3560.4.3.5, wherein the effectiveconcentration of at least the ALS inhibitor chemistry wouldsignificantly damage an appropriate control plant. In one non-limitingembodiment, the herbicide comprises at least one of asulfonylaminocarbonyltriazolinone; a triazolopyrimidine; apyrimidinyl(thio)benzoate; an imidazolinone; a triazine; and/or aphosphinic acid.

In another non-limiting embodiment, the combination of herbicidescomprises glyphosate, imazapyr, chlorimuron-ethyl, quizalofop, andfomesafen, wherein an effective amount is tolerated by the crop andcontrols weeds. As disclosed elsewhere herein, any effective amount ofthese herbicides can be applied. In specific embodiments, thiscombination of herbicides comprises an effective amount of glyphosatecomprising about 1110 to about 1130 g ai/hectare; an effective amount ofimazapyr comprising about 7.5 to about 27.5 g ai/hectare; an effectiveamount of chlorimuron-ethyl comprising about 7.5 to about 27.5 gai/hectare; an effective amount of quizalofop comprising about 50 toabout 70 g ai/hectare; and, an effective amount of fomesafen comprisingabout 240 to about 260 g ai/hectare.

In other embodiments, a combination of at least two herbicides isapplied, wherein the combination does not include glyphosate. In otherembodiments, at least one ALS inhibitor and glyphosate is applied to theplant. More details regarding the various herbicide combinations thatcan be employed in the methods are discussed elsewhere herein.

In one embodiment, the method of controlling weeds comprises plantingthe area with the 3560.4.3.5 soybean seeds or plants and applying to thecrop, crop part, seed of said crop or the area under cultivation, aneffective amount of a herbicide, wherein said effective amount comprises

i) an amount that is not tolerated by a first control crop when appliedto the first control crop, crop part, seed or the area of cultivation,wherein said first control crop expresses a first polynucleotideencoding GLYAT polypeptide that confers tolerance to glyphosate and doesnot express a second polynucleotide that encodes the gm-hra polypeptide;

ii) an amount that is not tolerated by a second control crop whenapplied to the second crop, crop part, seed or the area of cultivation,wherein said second control crop expresses the gm-hra polynucleotide anddoes not express the glyat polynucleotide; and,

iii) an amount that is tolerated when applied to the 3560.4.3.5 soybeancrop, crop part, seed, or the area of cultivation thereof. The herbicidecan comprise a combination of herbicides that either includes or doesnot include glyphosate. In specific embodiments, the combination ofherbicides comprises ALS inhibitor chemistries as discussed in furtherdetail below.

In another embodiment, the method of controlling weeds comprisesplanting the area with a 3560.4.3.5 soybean crop seed or plant andapplying to the crop, crop part, seed of said crop or the area undercultivation, an effective amount of a herbicide, wherein said effectiveamount comprises a level that is above the recommended label use ratefor the crop, wherein said effective amount is tolerated when applied tothe 3560.4.3.5 soybean crop, crop part, seed, or the area of cultivationthereof. The herbicide applied can comprise a combination of herbicidesthat either includes or does not include glyphosate. In specificembodiments, the combination of herbicides comprises at least one ALSinhibitor chemistry as discussed in further detail below. Furtherherbicides and combinations thereof that can be employed in the variousmethods are discussed in further detail below.

A “control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of the subject plantor plant cell, and may be any suitable plant or plant cell. A controlplant or plant cell may comprise, for example: (a) a wild-type plant orcell, i.e., of the same genotype as the starting material for thegenetic alteration which resulted in the subject plant or cell; (b) aplant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell which is genetically identical to thesubject plant or plant cell but which is not exposed to the sametreatment (e.g., herbicide treatment) as the subject plant or plantcell; (e) the subject plant or plant cell itself, under conditions inwhich the gene of interest is not expressed; or (f) the subject plant orplant cell itself, under conditions in which it has not been exposed toa particular treatment such as, for example, a herbicide or combinationof herbicides and/or other chemicals. In some instances, an appropriatecontrol plant or control plant cell may have a different genotype fromthe subject plant or plant cell but may share the herbicide-sensitivecharacteristics of the starting material for the genetic alteration(s)which resulted in the subject plant or cell (see, e.g., Green (1998)Weed Technology 12: 474-477; Green and Ulrich (1993) Weed Science 41:508-516. In some instances, an appropriate control soybean plant is a“Jack” soybean plant (Illinois Foundation Seed, Champaign, Ill.). Inother embodiments, the null segregant can be used as a control, as theyare near isogenic to 3560.4.3.5 with the exception of the transgenicinsert DNA.

Any herbicide can be applied to the 3560.4.3.5 soybean crop, crop part,or the area of cultivation containing the crop plant. Classification ofherbicides (i.e., the grouping of herbicides into classes andsubclasses) is well-known in the art and includes classifications byHRAC (Herbicide Resistance Action Committee) and WSSA (the Weed ScienceSociety of America) (see also, Retzinger and Mallory-Smith (1997) WeedTechnology 11: 384-393). An abbreviated version of the HRACclassification (with notes regarding the corresponding WSSA group) isset forth below in Table 1.

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., preemergent or postemergent), by the method of application (e.g.,foliar application or soil application), or by how they are taken up byor affect the plant. For example, thifensulfuron-methyl andtribenuron-methyl are applied to the foliage of a crop and are generallymetabolized there, while rimsulfuron and chlorimuron-ethyl are generallytaken up through both the roots and foliage of a plant. “Mode of action”generally refers to the metabolic or physiological process within theplant that the herbicide inhibits or otherwise impairs, whereas “site ofaction” generally refers to the physical location or biochemical sitewithin the plant where the herbicide acts or directly interacts.Herbicides can be classified in various ways, including by mode ofaction and/or site of action (see, e.g., Table 1).

Often, an herbicide-tolerance gene that confers tolerance to aparticular herbicide or other chemical on a plant expressing it willalso confer tolerance to other herbicides or chemicals in the same classor subclass, for example, a class or subclass set forth in Table 1.Thus, in some embodiments, a transgenic plant is tolerant to more thanone herbicide or chemical in the same class or subclass, such as, forexample, an inhibitor of PPO, a sulfonylurea, or a synthetic auxin.

Typically, the plants can tolerate treatment with different types ofherbicides (i.e., herbicides having different modes of action and/ordifferent sites of action) as well as with higher amounts of herbicidesthan previously known plants, thereby permitting improved weedmanagement strategies that are recommended in order to reduce theincidence and prevalence of herbicide-tolerant weeds. Specific herbicidecombinations can be employed to effectively control weeds.

Transgenic soybean plants are provided which can be selected for use incrop production based on the prevalence of herbicide-tolerant weedspecies in the area where the transgenic crop is to be grown. Weedmanagement techniques, such as for example, crop rotation using a cropthat is tolerant to a herbicide to which the local weed species are nottolerant can be used. See, for example, the Herbicide Resistance ActionCommittee (HRAC), the Weed Science Society of America, and various stateagencies and the herbicide tolerance scores for various broadleaf weedsfrom the 2004 Illinois Agricultural Pest Management Handbook). See also,Owen and Hartzler (2004), 2005 Herbicide Manual for AgriculturalProfessionals, Pub. WC 92 Revised (Iowa State University Extension, IowaState University of Science and Technology, Ames, Iowa); Weed Controlfor Corn, Soybeans, and Sorghum, Chapter 2 of “2004 IllinoisAgricultural Pest Management Handbook” (University of IllinoisExtension, University of Illinois at Urbana-Champaign, Illinois); WeedControl Guide for Field Crops, MSU Extension Bulletin E434 (MichiganState University, East Lansing, Mich.)).

TABLE 1 Abbreviated version of HRAC Herbicide Classification I. ALSInhibitors (WSSA Group 2) A. Sulfonylureas 1. Azimsulfuron 2.Chlorimuron-ethyl 3. Metsulfuron-methyl 4. Nicosulfuron 5. Rimsulfuron6. Sulfometuron-methyl 7. Thifensulfuron-methyl 8. Tribenuron-methyl 9.Amidosulfuron 10. Bensulfuron-methyl 11. Chlorsulfuron 12. Cinosulfuron13. Cyclosulfamuron 14. Ethametsulfuron-methyl 15. Ethoxysulfuron 16.Flazasulfuron 17. Flupyrsulfuron-methyl 18. Foramsulfuron 19.Imazosulfuron 20. Iodosulfuron-methyl 21. Mesosulfuron-methyl 22.Oxasulfuron 23. Primisulfuron-methyl 24. Prosulfuron 25.Pyrazosulfuron-ethyl 26. Sulfosulfuron 27. Triasulfuron 28.Trifloxysulfuron 29. Triflusulfuron-methyl 30. Tritosulfuron 31.Halosulfuron-methyl 32. Flucetosulfuron B.Sulfonylaminocarbonyltriazolinones 1. Flucarbazone 2. Procarbazone C.Triazolopyrimidines 1. Cloransulam-methyl 2. Flumetsulam 3. Diclosulam4. Florasulam 5. Metosulam 6. Penoxsulam 7. Pyroxsulam D.Pyrimidinyloxy(thio)benzoates 1. Bispyribac 2. Pyriftalid 3.Pyribenzoxim 4. Pyrithiobac 5. Pyriminobac-methyl E. Imidazolinones 1.Imazapyr 2. Imazethapyr 3. Imazaquin 4. Imazapic 5.Imazamethabenz-methyl 6. Imazamox II. Other Herbicides - ActiveIngredients/Additional Modes of Action A. Inhibitors of Acetyl CoAcarboxylase (ACCase) (WSSA Group 1) 1. Aryloxyphenoxypropionates(‘FOPs’) a. Quizalofop-P-ethyl b. Diclofop-methyl c.Clodinafop-propargyl d. Fenoxaprop-P-ethyl e. Fluazifop-P-butyl f.Propaquizafop g. Haloxyfop-P-methyl h. Cyhalofop-butyl i.Quizalofop-P-ethyl 2. Cyclohexanediones (‘DIMs’) a. Alloxydim b.Butroxydim c. Clethodim d. Cycloxydim e. Sethoxydim f. Tepraloxydim g.Tralkoxydim B. Inhibitors of Photosystem II - HRAC Group C1/WSSA Group5 1. Triazines a. Ametryne b. Atrazine c. Cyanazine d. Desmetryne e.Dimethametryne f. Prometon g. Prometryne h. Propazine i. Simazine j.Simetryne k. Terbumeton l. Terbuthylazine m. Terbutryne n. Trietazine 2.Triazinones a. Hexazinone b. Metribuzin c. Metamitron 3. Triazolinone a.Amicarbazone 4. Uracils a. Bromacil b. Lenacil c. Terbacil 5.Pyridazinones a. Pyrazon 6. Phenyl carbamates a. Desmedipham b.Phenmedipham C. Inhibitors of Photosystem II - HRAC Group C2/WSSA Group7 1. Ureas a. Fluometuron b. Linuron c. Chlorobromuron d. Chlorotolurone. Chloroxuron f. Dimefuron g. Diuron h. Ethidimuron i. Fenuron j.Isoproturon k. Isouron l. Methabenzthiazuron m. Metobromuron n.Metoxuron o. Monolinuron p. Neburon q. Siduron r. Tebuthiuron 2. Amidesa. Propanil b. Pentanochlor D. Inhibitors of Photosystem II - HRAC GroupC3/WSSA Group 6 1. Nitriles a. Bromofenoxim b. Bromoxynil c. Ioxynil 2.Benzothiadiazinone (Bentazon) a. Bentazon 3. Phenylpyridazines a.Pyridate b. Pyridafol E. Photosystem-I-electron diversion(Bipyridyliums) (WSSA Group 22) 1. Diquat 2. Paraquat F. Inhibitors ofPPO (protoporphyrinogen oxidase) (WSSA Group 14) 1. Diphenylethers a.Acifluorfen-Na b. Bifenox c. Chlomethoxyfen d. Fluoroglycofen-ethyl e.Fomesafen f. Halosafen g. Lactofen h. Oxyfluorfen 2. Phenylpyrazoles a.Fluazolate b. Pyraflufen-ethyl 3. N-phenylphthalimides a. Cinidon-ethylb. Flumioxazin c. Flumiclorac-pentyl 4. Thiadiazoles a.Fluthiacet-methyl b. Thidiazimin 5. Oxadiazoles a. Oxadiazon b.Oxadiargyl 6. Triazolinones a. Carfentrazone-ethyl b. Sulfentrazone 7.Oxazolidinediones a. Pentoxazone 8. Pyrimidindiones a. Benzfendizone b.Butafenicil 9. Others a. Pyrazogyl b. Profluazol G. Bleaching:Inhibition of carotenoid biosynthesis at the phytoene desaturase step(PDS) (WSSA Group 12) 1. Pyridazinones a. Norflurazon 2.Pyridinecarboxamides a. Diflufenican b. Picolinafen 3. Others a.Beflubutamid b. Fluridone c. Flurochloridone d. Flurtamone H. Bleaching:Inhibition of 4-hydroxyphenyl-pyruvate-dioxygenase (4-HPPD) (WSSA Group28) 1. Triketones a. Mesotrione b. Sulcotrione 2. Isoxazoles a.Isoxachlortole b. Isoxaflutole 3. Pyrazoles a. Benzofenap b. Pyrazoxyfenc. Pyrazolynate 4. Others a. Benzobicyclon I. Bleaching: Inhibition ofcarotenoid biosynthesis (unknown target) (WSSA Group 11 and 13) 1.Triazoles (WSSA Group 11) a. Amitrole 2. Isoxazolidinones (WSSA Group13) a. Clomazone 3. Ureas a. Fluometuron 3. Diphenylether a. AclonifenJ. Inhibition of EPSP Synthase 1. Glycines (WSSA Group 9) a. Glyphosateb. Sulfosate K. Inhibition of glutamine synthetase 1. Phosphinic Acidsa. Glufosinate-ammonium b. Bialaphos L. Inhibition of DHP(dihydropteroate) synthase (WSSA Group 18) 1 Carbamates a. Asulam M.Microtubule Assembly Inhibition (WSSA Group 3) 1. Dinitroanilines a.Benfluralin b. Butralin c. Dinitramine d. Ethalfluralin e. Oryzalin f.Pendimethalin g. Trifluralin 2. Phosphoroamidates a. Amiprophos-methylb. Butamiphos 3. Pyridines a. Dithiopyr b. Thiazopyr 4. Benzamides a.Pronamide b. Tebutam 5. Benzenedicarboxylic acids a. Chlorthal-dimethylN. Inhibition of mitosis/microtubule organization WSSA Group 23) 1.Carbamates a. Chlorpropham b. Propham c. Carbetamide O. Inhibition ofcell division (Inhibition of very long chain fatty acids as proposedmechanism; WSSA Group 15) 1. Chloroacetamides a. Acetochlor b. Alachlorc. Butachlor d. Dimethachlor e. Dimethanamid f. Metazachlor g.Metolachlor h. Pethoxamid i. Pretilachlor j. Propachlor k. Propisochlorl. Thenylchlor 2. Acetamides a. Diphenamid b. Napropamide c.Naproanilide 3. Oxyacetamides a. Flufenacet b. Mefenacet 4.Tetrazolinones a. Fentrazamide 5. Others a. Anilofos b. Cafenstrole c.Indanofan d. Piperophos P. Inhibition of cell wall (cellulose)synthesis 1. Nitriles (WSSA Group 20) a. Dichlobenil b. Chlorthiamid 2.Benzamides (isoxaben (WSSA Group 21)) a. Isoxaben 3.Triazolocarboxamides (flupoxam) a. Flupoxam Q. Uncoupling (membranedisruption): (WSSA Group 24) 1. Dinitrophenols a. DNOC b. Dinoseb c.Dinoterb R. Inhibition of Lipid Synthesis by other than ACCinhibition 1. Thiocarbamates (WSSA Group 8) a. Butylate b. Cycloate c.Dimepiperate d. EPTC e. Esprocarb f. Molinate g. Orbencarb h. Pebulatei. Prosulfocarb j. Benthiocarb k. Tiocarbazil l. Triallate m. Vernolate2. Phosphorodithioates a. Bensulide 3. Benzofurans a. Benfuresate b.Ethofumesate 4. Halogenated alkanoic acids (WSSA Group 26) a. TCA b.Dalapon c. Flupropanate S. Synthetic auxins (IAA-like) (WSSA Group 4) 1.Phenoxycarboxylic acids a. Clomeprop b. 2,4-D c. Mecoprop 2. Benzoicacids a. Dicamba b. Chloramben c. TBA 3. Pyridine carboxylic acids a.Clopyralid b. Fluroxypyr c. Picloram d. Tricyclopyr 4. Quinolinecarboxylic acids a. Quinclorac b. Quinmerac 5. Others (benazolin-ethyl)a. Benazolin-ethyl T. Inhibition of Auxin Transport 1. Phthalamates;semicarbazones (WSSA Group 19) a. Naptalam b. Diflufenzopyr-Na U. OtherMechanism of Action 1. Arylaminopropionic acids a.Flamprop-M-methyl/-isopropyl 2. Pyrazolium a. Difenzoquat 3.Organoarsenicals a. DSMA b. MSMA 4. Others a. Bromobutide b. Cinmethylinc. Cumyluron d. Dazomet e. Daimuron-methyl f. Dimuron g. Etobenzanid h.Fosamine i. Metam j. Oxaziclomefone k. Oleic acid l. Pelargonic acid m.Pyributicarb

In one embodiment, one ALS inhibitor or at least two ALS inhibitors areapplied to the 3560.4.3.5 soybean crop or area of cultivation. Innon-limiting embodiments, the combination of ALS inhibitor herbicidescan include or does not include glyphosate. The ALS inhibitor can beapplied at any effective rate that selectively controls weeds and doesnot significantly damage the crop. In specific embodiments, at least oneALS inhibitor is applied at a level that would significantly damage anappropriate control plant. In other embodiments, at least one ALSinhibitor is applied above the recommended label use rate for the crop.In still other embodiments, a mixture of ALS inhibitors is applied at alower rate than the recommended use rate and weeds continue to beselectively controlled. Herbicides that inhibit acetolactate synthase(also known as acetohydroxy acid synthase) and are therefore useful inthe methods include sulfonylureas as listed in Table 1, includingagriculturally suitable salts (e.g., sodium salts) thereof;sulfonylaminocarbonyltriazolinones as listed in Table 1, includingagriculturally suitable salts (e.g., sodium salts) thereof;triazolopyrimidines as listed in Table 1, including agriculturallysuitable salts (e.g., sodium salts) thereof;pyrimidinyloxy(thio)benzoates as listed in Table 1, includingagriculturally suitable salts (e.g., sodium salts) thereof; andimidazolinones as listed in Table 1, including agriculturally suitablesalts (e.g., sodium salts) thereof. In some embodiments, methodscomprise the use of a sulfonylurea which is not chlorimuron-ethyl,chlorsulfuron, rimsulfuron, thifensulfuron-methyl, or tribenuron-methyl.

In still further methods, glyphosate, alone or in combination withanother herbicide of interest, can be applied to the 3560.4.3.5 soybeanplants or their area of cultivation. Non-limiting examples of glyphosateformations are set forth in Table 2. In specific embodiments, theglyphosate is in the form of a salt, such as, ammonium,isopropylammonium, potassium, sodium (including sesquisodium) ortrimesium (alternatively named sulfosate). In still further embodiments,a mixture of a synergistically effective amount of a combination ofglyphosate and an ALS inhibitor (such as a sulfonylurea) is applied tothe 3560.4.3.5 soybean plants or their area of cultivation.

TABLE 2 Glyphosate formulations comparisons. Active Acid Acid ingredientequivalent Apply: equivalent Herbicide by Registered per per # oz/ perTrademark Manufacturer Salt gallon gallon acre acre Roundup OriginalMonsanto Isopropylamine 4 3 32 0.750 Roundup Original II MonsantoIsopropylamine 4 3 32 0.750 Roundup Original Max Monsanto Potassium 5.54.5 22 0.773 Roundup UltraMax Monsanto Isopropylamine 5 3.68 26 0.748Roundup UltraMax II Monsanto Potassium 5.5 4.5 22 0.773 RoundupWeathermax Monsanto Potassium 5.5 4.5 22 0.773 Touchdown SyngentaDiammomium 3.7 3 32 0.750 Touchdown HiTech Syngenta Potassium 6.16 5 200.781 Touchdown Total Syngenta Potassium 5.14 4.17 24 0.782 Durango DowAgroSciences Isopropylamine 5.4 4 24 0.750 Glyphomax Dow AgroSciencesIsopropylamine 4 3 32 0.750 Glyphomax Plus Dow AgroSciencesIsopropylamine 4 3 32 0.750 Glyphomax XRT Dow AgroSciencesIsopropylamine 4 3 32 0.750 Gly Star Plus Albaugh/Agri StarIsopropylamine 4 3 32 0.750 Gly Star 5 Albaugh/Agri Star Isopropylamine5.4 4 24 0.750 Gly Star Original Albaugh/Agri Star Isopropylamine 4 3 320.750 Gly-Flo Micro Flo Isopropylamine 4 3 32 0.750 Credit NufarmIsopropylamine 4 3 32 0.750 Credit Extra Nufarm Isopropylamine 4 3 320.750 Credit Duo Nufarm Isopro. + 4 3 32 0.750 monoamm. Credit Duo ExtraNufarm Isopro. + 4 3 32 0.750 monoamm. Extra Credit 5 NufarmIsopropylamine 5 3.68 26 0.748 Comerstone Agriliance Isopropylamine 4 332 0.750 Comerstone Plus Agriliance Isopropylamine 4 3 32 0.750 GlyfosCheminova Isopropylamine 4 3 32 0.750 Glyfos X-TRA CheminovaIsopropylamine 4 3 32 0.750 Rattier Helena Isopropylamine 4 3 32 0.750Rattier Plus Helena Isopropylamine 4 3 32 0.750 Mirage UAPIsopropylamine 4 3 32 0.750 Mirage Plus UAP Isopropylamine 4 3 32 0.750Glyphosate 41% Halm Agro USA Isopropylamine 4 3 32 0.750 BuccaneerTenkoz Isopropylamine 4 3 32 0.750 Buccaneer Plus Tenkoz Isopropylamine4 3 32 0.750 Honcho Monsanto Isopropylamine 4 3 32 0.750 Honcho PlusMonsanto Isopropylamine 4 3 32 0.750 Gly-4 Univ. Crop Prot. Alli.Isopropylamine 4 3 32 0.750 Gly-4 Plus Univ. Crop Prot. Alli.Isopropylamine 4 3 32 0.750 ClearOut 41 Chemical Products Isopropylamine4 3 32 0.750 Tech. ClearOut 41 Plus Chemical Products Isopropylamine 4 332 0.750 Tech. Spitfire Control Soultions Isopropylamine 4 3 32 0.750Spitfire Plus Control Soultions Isopropylamine 4 3 32 0.750 Glyphosate 4FarmerSaver.com Isopropylamine 4 3 32 0.750 FS Glyphosate Plus GrowmarkIsopropylamine 4 3 32 0.750 Glyphosate Original Griffin, LLC.Isopropylamine 4 3 32 0.750

Thus, in some embodiments, a transgenic plant is used in a method ofgrowing a 3560.4.3.5 soybean crop by the application of herbicides towhich the plant is tolerant. In this manner, treatment with acombination of one of more herbicides which include, but are not limitedto: acetochlor, acifluorfen and its sodium salt, aclonifen, acrolein(2-propenal), alachlor, alloxydim, ametryn, amicarbazone, amidosulfuron,aminopyralid, amitrole, ammonium sulfamate, anilofos, asulam, atrazine,azimsulfuron, beflubutamid, benazolin, benazolin-ethyl, bencarbazone,benfluralin, benfuresate, bensulfuron-methyl, bensulide, bentazone,benzobicyclon, benzofenap, bifenox, bilanafos, bispyribac and its sodiumsalt, bromacil, bromobutide, bromofenoxim, bromoxynil, bromoxyniloctanoate, butachlor, butafenacil, butamifos, butralin, butroxydim,butylate, cafenstrole, carbetamide, carfentrazone-ethyl, catechin,chlomethoxyfen, chloramben, chlorbromuron, chlorflurenol-methyl,chloridazon, chlorimuron-ethyl, chlorotoluron, chlorpropham,chlorsulfuron, chlorthal-dimethyl, chlorthiamid, cinidon-ethyl,cinmethylin, cinosulfuron, clethodim, clodinafop-propargyl, clomazone,clomeprop, clopyralid, clopyralid-olamine, cloransulam-methyl, CUH-35(2-methoxyethyl2-[[[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl](3-fluorobenzoyl)amino]carbonyl]-1-cyclohexene-1-carboxylate),cumyluron, cyanazine, cycloate, cyclosulfamuron, cycloxydim,cyhalofop-butyl, 2,4-D and its butotyl, butyl, isoctyl and isopropylesters and its dimethylammonium, diolamine and trolamine salts,daimuron, dalapon, dalapon-sodium, dazomet, 2,4-DB and itsdimethylammonium, potassium and sodium salts, desmedipham, desmetryn,dicamba and its diglycolammonium, dimethylammonium, potassium and sodiumsalts, dichlobenil, dichlorprop, diclofop-methyl, diclosulam,difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron,dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P,dimethipin, dimethylarsinic acid and its sodium salt, dinitramine,dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC,endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl,ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-ethyl,fenoxaprop-P-ethyl, fentrazamide, fenuron, fenuron-TCA, flamprop-methyl,flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam,fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucetosulfuron,fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam,flumiclorac-pentyl, flumioxazine, fluometuron, fluoroglycofen-ethyl,flupyrsulfuron-methyl and its sodium salt, flurenol, flurenol-butyl,fluridone, fluorochloridone, fluoroxypyr, flurtamone, fluthiacet-methyl,fomesafen, foramsulfuron, fosamine-ammonium, glufosinate,glufosinate-ammonium, glyphosate and its salts such as ammonium,isopropylammonium, potassium, sodium (including sesquisodium) andtrimesium (alternatively named sulfosate), halosulfuron-methyl,haloxyfop-etotyl, haloxyfop-methyl, hexazinone, HOK-201(N-(2,4-difluorophenyl)-1,5-dihydro-N-(1-methylethyl)-5-oxo-1-[(tetrahydro-2H-pyran-2-yl)methyl]-4H-1,2,4-triazole-4-carboxamide),imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin,imazaquin-ammonium, imazethapyr, imazethapyr-ammonium, imazosulftiron,indanofan, iodosulfuron-methyl, ioxynil, ioxynil octanoate,ioxynil-sodium, isoproturon, isouron, isoxaben, isoxaflutole,isoxachlortole, lactofen, lenacil, linuron, maleic hydrazide, MCPA andits salts (e.g., MCPA-dimethylammonium, MCPA-potassium and MCPA-sodium,esters (e.g., MCPA-2-ethylhexyl, MCPA-butotyl) and thioesters (e.g.,MCPA-thioethyl), MCPB and its salts (e.g., MCPB-sodium) and esters(e.g., MCPB-ethyl), mecoprop, mecoprop-P, mefenacet, mefluidide,mesosulfuron-methyl, mesotrione, metam-sodium, metamifop, metamitron,metazachlor, methabenzthiazuron, methylarsonic acid and its calcium,monoammonium, monosodium and disodium salts, methyldymron, metobenzuron,metebromuron, metolachlor, S-metholachlor, metosulam, metoxuron,metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide,napropamide, naptalam, neburon, nicosulfuron, norflurazon, orbencarb,oryzalin, oxadiargyl, oxadiazon, oxasulfinuron, oxaziclomefone,oxyfluorfen, paraquat dichloride, pebulate, pelargonic acid,pendamethalin, penoxsulam, pentanochlor, pentoxazone, perfluidone,pethoxyamid, phenmedipham, picloram, picloram-potassium, picolinafen,pinoxaden, piperofos, pretilachlor, primisulfuron-methyl, prodiamine,profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop,propazine, propham, propisochlor, propoxycarbazone, propyzamide,prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl, pyrasulfotole,pyrazogyl, pyrazolynate, pyrazoxyfen, pyrazosulfuron-ethyl,pyribenzoxim, pyributicarb, pyridate, pyriftalid, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxsulam, quinclorac,quinmerac, quinoclamine, quizalofop-ethyl, quizalofop-P-ethyl,quizalofop-P-tefuryl, rimsulfuron, sethoxydim, siduron, simazine,simetryn, sulcotrione, sulfentrazone, sulfometuron-methyl,sulfosulfuron, 2,3,6-TBA, TCA, TCA-sodium, tebutam, tebuthiuron,tefuryltrione, tembotrione, tepraloxydim, terbacil, terbumeton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thiencarbazone,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl,triclopyr, triclopyr-butotyl, triclopyr-triethylammonium, tridiphane,trietazine, trifloxysulfuron, trifluralin, triflusulfuron-methyl,tritosulfuron and vernolate is disclosed.

Other suitable herbicides and agricultural chemicals are known in theart, such as, for example, those described in WO 2005/041654. Otherherbicides also include bioherbicides such as Alternaria destruensSimmons, Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc., Drechsieramonoceras (MTB-951), Myrothecium verrucaria (Albertini & Schweinitz)Ditmar: Fries, Phytophthora palmivora (Butyl.) Butyl. and Pucciniathlaspeos Schub. Combinations of various herbicides can result in agreater-than-additive (i.e., synergistic) effect on weeds and/or aless-than-additive effect (i.e. safening) on crops or other desirableplants. In certain instances, combinations of glyphosate with otherherbicides having a similar spectrum of control but a different mode ofaction will be particularly advantageous for preventing the developmentof resistant weeds. Herbicidally effective amounts of any particularherbicide can be easily determined by one skilled in the art throughsimple experimentation.

Herbicides may be classified into groups and/or subgroups as describedherein above with reference to their mode of action, or they may beclassified into groups and/or subgroups in accordance with theirchemical structure.

Sulfonamide herbicides have as an essential molecular structure featurea sulfonamide moiety (—S(O)₂NH—). As referred to herein, sulfonamideherbicides particularly comprise sulfonylurea herbicides,sulfonylaminocarbonyltriazolinone herbicides and triazolopyrimidineherbicides. In sulfonylurea herbicides the sulfonamide moiety is acomponent in a sulfonylurea bridge (—S(O)₂NHC(O)NH(R)—). In sulfonylureaherbicides the sulfonyl end of the sulfonylurea bridge is connectedeither directly or by way of an oxygen atom or an optionally substitutedamino or methylene group to a typically substituted cyclic or acyclicgroup. At the opposite end of the sulfonylurea bridge, the amino group,which may have a substituent such as methyl (R being CH₃) instead ofhydrogen, is connected to a heterocyclic group, typically a symmetricpyrimidine or triazine ring, having one or two substituents such asmethyl, ethyl, trifluoromethyl, methoxy, ethoxy, methylamino,dimethylamino, ethylamino and the halogens. Insulfonylaminocarbonyltriazolinone herbicides, the sulfonamide moiety isa component of a sulfonylaminocarbonyl bridge (—S(O)₂NHC(O)—). Insulfonylamino-carbonyltriazolinone herbicides the sulfonyl end of thesulfonylaminocarbonyl bridge is typically connected to substitutedphenyl ring. At the opposite end of the sulfonylaminocarbonyl bridge,the carbonyl is connected to the 1-position of a triazolinone ring,which is typically substituted with groups such as alkyl and alkoxy. Intriazolopyrimidine herbicides the sulfonyl end of the sulfonamide moietyis connected to the 2-position of a substituted[1,2,4]triazolopyrimidine ring system and the amino end of thesulfonamide moiety is connected to a substituted aryl, typically phenyl,group or alternatively the amino end of the sulfonamide moiety isconnected to the 2-position of a substituted [1,2,4]triazolopyrimidinering system and the sulfonyl end of the sulfonamide moiety is connectedto a substituted aryl, typically pyridinyl, group.

Representative of the sulfonylurea herbicides useful in the embodimentsare those of the formula:

wherein:

J is selected from the group consisting of

-   -   J is R¹³SO₂N(CH₃)—;    -   R is H or CH₃;    -   R¹ is F, Cl, Br, NO₂, C₁-C₄ alkyl, C₁-C₄ haloalkyl, C₃-C₄        cycloalkyl, C₂-C₄ haloalkenyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy,        C₂-C₄ alkoxyalkoxy, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰, CH₂CN or L;    -   R² is H, F, Cl, Br, I, CN, CH₃, OCH₃, SCH₃, CF₃ or OCF₂H;    -   R³ is Cl, NO₂, CO₂CH₃, CO₂CH₂CH₃, C(O)CH₃, C(O)CH₂CH₃,        C(O)-cyclopropyl, SO₂N(CH₃)₂, SO₂CH₃, SO₂CH₂CH₃, OCH₃ or        OCH₂CH₃;    -   R⁴ is C₁-C₃ alkyl, C₁-C₂ haloalkyl, C₁-C₂ alkoxy, C₂-C₄        haloalkenyl, F, Cl, Br, NO₂, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R⁵ is H, F, Cl, Br or CH₃;    -   R⁶ is C₁-C₃ alkyl optionally substituted with 0-3 F, 0-1 Cl and        0-1 C₃-C₄ alkoxyacetyloxy, or R⁶ is C₁-C₂ alkoxy, C₂-C₄        haloalkenyl, F, Cl, Br, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸,        S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R⁷ is H, F, Cl, CH₃ or CF₃;    -   R⁸ is H, C₁-C₃ alkyl or pyridinyl;    -   R⁹ is C₁-C₃alkyl, C₁-C₂ alkoxy, F, Cl, Br, NO₂, CO₂R¹⁴,        SO₂NR¹⁷R¹⁸, S(O)_(n)R¹⁹, OCF₂H, C(O)R²⁰, C₂-C₄ haloalkenyl or L;    -   R¹⁰ is H, Cl, F, Br, C₁-C₃ alkyl or C₁-C₂ alkoxy;    -   R¹¹ is H, C₁-C₃ alkyl, C₁-C₂ alkoxy, C₂-C₄ haloalkenyl, F, Cl,        Br, CO₂R¹⁴, C(O)NR¹⁵R¹⁶, SO₂NR¹⁷R¹⁸, S(O)_(n)R¹⁹, C(O)R²⁰ or L;    -   R¹² is halogen, C₁-C₄ alkyl or C₁-C₃ alkylsulfonyl;    -   R¹³ is C₁-C₄ alkyl;    -   R¹⁴ is allyl, propargyl or oxetan-3-yl; or R¹⁴ is C₁-C₃ alkyl        optionally substituted by at least one member independently        selected from halogen, C₁-C₂ alkoxy and CN;    -   R¹⁵ is H, C₁-C₃ alkyl or C₁-C₂ alkoxy;    -   R¹⁶ is C₁-C₂ alkyl;    -   R¹⁷ is H, C₁-C₃ alkyl, C₁-C₂ alkoxy, allyl or cyclopropyl;    -   R¹⁸ is H or C₁-C₃ alkyl;    -   R¹⁹ is C₁-C₃ alkyl, C₁-C₃ haloalkyl, allyl or propargyl;    -   R²⁰ is C₁-C₄ alkyl, C₁-C₄ haloalkyl or C₃-C₅ cycloalkyl        optionally substituted by halogen;    -   n is 0, 1 or 2;    -   L is

-   -   L¹ is CH₂, NH or O;    -   R²¹ is H or C₁-C₃ alkyl;    -   X is H, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₁-C₄        haloalkyl, C₁-C₄ haloalkylthio, C₁-C₄ alkylthio, halogen, C₂-C₅        alkoxyalkyl, C₂-C₅ alkoxyalkoxy, amino, C₁-C₃ alkylamino or        di(C₁-C₃ alkyl)amino;    -   Y is H, C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₁-C₄        alkylthio, C₁-C₄ haloalkylthio, C₂-C₅ alkoxyalkyl, C₂-C₅        alkoxyalkoxy, amino, C₁-C₃ alkylamino, di(C₁-C₃ alkyl)amino,        C₃-C₄ alkenyloxy, C₃-C₄ alkynyloxy, C₂-C₅ alkylthioalkyl, C₂-C₅        alkylsulfinylalkyl, C₂-C₅ alkylsulfonylalkyl, C₁-C₄ haloalkyl,        C₂-C₄ alkynyl, C₃-C₅ cycloalkyl, azido or cyano; and    -   Z is CH or N;

provided that (i) when one or both of X and Y is C₁ haloalkoxy, then Zis CH; and (ii) when X is halogen, then Z is CH and Y is OCH₃, OCH₂CH₃,N(OCH₃)CH₃, NHCH₃, N(CH₃)₂ or —OCF₂H. Of note is the present singleliquid herbicide composition comprising one or more sulfonylureas ofFormula I wherein when R⁶ is alkyl, said alkyl is unsubstituted.

Representative of the triazolopyrimidine herbicides contemplated for usein the embodiments are those of the formula:

wherein:

-   -   R²² and R²³ each independently halogen, nitro, C₁-C₄ alkyl,        C₁-C₄ haloalkyl, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy or C₂-C₃        alkoxycarbonyl;    -   R²⁴ is H, halogen, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   W is —NHS(O)₂— or —S(O)₂NH—;    -   Y¹ is H, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   Y² is H, F, Cl, Br, C₁-C₂ alkyl or C₁-C₂ alkoxy;    -   Y³ is H, F or methoxy;    -   Z¹ is CH or N; and    -   Z² is CH or N;        provided that at least one of Y¹ and Y² is other than H.

In the above Markush description of representative triazolopyrimidineherbicides, when W is —NHS(O)₂— the sulfonyl end of the sulfonamidemoiety is connected to the [1,2,4]triazolopyrimidine ring system, andwhen W is —S(O)₂NH— the amino end of the sulfonamide moiety is connectedto the [1,2,4]triazolopyrimidine ring system.

In the above recitations, the term “alkyl”, used either alone or incompound words such as “alkylthio” or “haloalkyl” includesstraight-chain or branched alkyl, such as, methyl, ethyl, n-propyl,i-propyl, or the different butyl isomers. “Cycloalkyl” includes, forexample, cyclopropyl, cyclobutyl and cyclopentyl. “Alkenyl” includesstraight-chain or branched alkenes such as ethenyl, 1-propenyl,2-propenyl, and the different butenyl isomers. “Alkenyl” also includespolyenes such as 1,2-propadienyl and 2,4-butadienyl. “Alkynyl” includesstraight-chain or branched alkynes such as ethynyl, 1-propynyl,2-propynyl and the different butynyl isomers. “Alkynyl” can also includemoieties comprised of multiple triple bonds such as 2,5-hexadienyl.“Alkoxy” includes, for example, methoxy, ethoxy, n-propyloxy,isopropyloxy and the different butoxy isomers. “Alkoxyalkyl” denotesalkoxy substitution on alkyl. Examples of “alkoxyalkyl” include CH₃OCH₂,CH₃OCH₂CH₂, CH₃CH₂OCH₂, CH₃CH₂CH₂CH₂OCH₂ and CH₃CH₂OCH₂CH₂.“Alkoxyalkoxy” denotes alkoxy substitution on alkoxy. “Alkenyloxy”includes straight-chain or branched alkenyloxy moieties. Examples of“alkenyloxy” include H₂C═CHCH₂O, (CH₃)CH═CHCH₂O and CH₂═CHCH₂CH₂O.“Alkynyloxy” includes straight-chain or branched alkynyloxy moieties.Examples of “alkynyloxy” include HC≡CCH₂O and CH₃C≡CCH₂O. “Alkylthio”includes branched or straight-chain alkylthio moieties such asmethylthio, ethylthio, and the different propylthio isomers.“Alkylthioalkyl” denotes alkylthio substitution on alkyl. Examples of“alkylthioalkyl” include CH₃SCH₂, CH₃SCH₂CH₂, CH₃CH₂SCH₂,CH₃CH₂CH₂CH₂SCH₂ and CH₃CH₂SCH₂CH₂; “alkylsulfinylalkyl” and“alkylsulfonyl-alkyl” include the corresponding sulfoxides and sulfones,respectively. Other substituents such as “alkylamino”, “dialkylamino”are defined analogously.

The total number of carbon atoms in a substituent group is indicated bythe “C_(i)-C_(j)” prefix where i and j are numbers from 1 to 5. Forexample, C₁-C₄ alkyl designates methyl through butyl, including thevarious isomers. As further examples, C₂ alkoxyalkyl designates CH₃OCH₂;C₃ alkoxyalkyl designates, for example, CH₃CH(OCH₃), CH₃OCH₂CH₂ orCH₃CH₂OCH₂; and C₄ alkoxyalkyl designates the various isomers of analkyl group substituted with an alkoxy group containing a total of fourcarbon atoms, examples including CH₃CH₂CH₂OCH₂ and CH₃CH₂OCH₂CH₂.

The term “halogen”, either alone or in compound words such as“haloalkyl”, includes fluorine, chlorine, bromine or iodine. Further,when used in compound words such as “haloalkyl”, said alkyl may bepartially or fully substituted with halogen atoms which may be the sameor different. Examples of “haloalkyl” include F₃C, ClCH₂, CF₃CH₂ andCF₃CCl₂. The terms “haloalkoxy”, “haloalkylthio”, and the like, aredefined analogously to the term “haloalkyl”. Examples of “haloalkoxy”include CF₃O, CCl₃CH₂O, HCF₂CH₂CH₂O and CF₃CH₂O. Examples of“haloalkylthio” include CCl₃S, CF₃S, CCl₃CH₂S and ClCH₂CH₂CH₂S.

The following sulfonylurea herbicides illustrate the sulfonylureasuseful for this invention: amidosulfuron(N-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]-sulfonyl]-N-methylmethanesulfonamide),azimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-1-methyl-4-(2-methyl-2H-tetrazol-5-yl)-1H-pyrazole-5-sulfonamide),bensulfuron-methyl (methyl2-[[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]methyl]benzoate),chlorimuron-ethyl (ethyl2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-benzoate),chlorsulfuron(2-chloro-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]-carbonyl]benzenesulfonamide),cinosulfuron(N-[[(4,6-dimethoxy-1,3,5-triazin-2-yl)amino]carbonyl]-2-(2-methoxyethoxy)benzenesulfonamide),cyclosulfamuron(N-[[[2-(cyclopropylcarbonyl)phenyl]amino]sulfonyl]-N¹-(4,6-dimethoxypyrimidin-2-yl)urea),ethametsulfuron-methyl (methyl2-[[[[[4-ethoxy-6-(methylamino)-1,3,5-triazin-2-yl]amino]carbonyl]amino]sulfonyl]benzoate),ethoxysulfuron (2-ethoxyphenyl[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]sulfamate), flazasulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(trifluoromethyl)-2-pyridinesulfonamide),flucetosulfuron(1-[3-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-2-pyridinyl]-2-fluoropropylmethoxyacetate), flupyrsulfuron-methyl (methyl2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-6-(trifluoromethyl)-3-pyridinecarboxylate),foramsulfuron(2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-4-(formylamino)-N,N-dimethylbenzamide),halosulfuron-methyl (methyl3-chloro-5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylate),imazosulfuron(2-chloro-N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]imidazo[1,2-a]pyridine-3-sulfonamide),iodosulfuron-methyl (methyl4-iodo-2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate),mesosulfuron-methyl (methyl2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-4-[[(methylsulfonyl)-amino]methyl]benzoate),metsulfuron-methyl (methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate),nicosulfuron(2-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-N,N-dimethyl-3-pyridinecarboxamide),oxasulfuron (3-oxetanyl2-[[[[(4,6-dimethyl-2-pyrimidinyl)-amino]carbonyl]amino]sulfonyl]benzoate),primisulfuron-methyl (methyl2-[[[[[4,6-bis(trifluoromethoxy)-2-pyrimidinyl]amino]carbonyl]amino]sulfonyl]benzoate),prosulfuron(N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]-2-(3,3,3-trifluoropropyl)benzenesulfonamide),pyrazosulfuron-ethyl (ethyl5-[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-1-methyl-1H-pyrazole-4-carboxylate),rimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide),sulfometuron-methyl (methyl2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]-benzoate),sulfosulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-2-(ethylsulfonyl)imidazo[1,2-a]pyridine-3-sulfonamide),thifensulfuron-methyl (methyl3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate),triasulfuron(2-(2-chloroethoxy)-N-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]benzenesulfonamide),tribenuron-methyl (methyl2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylamino]carbonyl]-amino]sulfonyl]benzoate),trifloxysulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]-carbonyl]-3-(2,2,2-trifluoroethoxy)-2-pyridinesulfonamide),triflusulfuron-methyl (methyl2-[[[[[4-dimethylamino)-6-(2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl]amino]-carbonyl]amino]sulfonyl]-3-methylbenzoate)and tritosulfuron(N-[[[4-methoxy-6-(trifluoromethyl)-1,3,5-triazin-2-yl]amino]carbonyl]-2-(trifluoromethyl)benzene-sulfonamide).

The following triazolopyrimidine herbicides illustrate thetriazolopyrimidines useful for this invention: cloransulam-methyl(methyl3-chloro-2-[[(5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)sulfonyl]amino]benzoate,diclosulam(N-(2,6-dichlorophenyl)-5-ethoxy-7-fluoro[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide,florasulam(N-(2,6-difluorophenyl)-8-fluoro-5-methoxy[1,2,4]triazolo[1,5-c]pyrimidine-2-sulfonamide),flumetsulam(N-(2,6-difluorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide),metosulam(N-(2,6-dichloro-3-methylphenyl)-5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide),penoxsulam(2-(2,2-difluoroethoxy)-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)-6-(trifluoromethyl)benzenesulfonamide)and pyroxsulam(N-(5,7-dimethoxy[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-2-methoxy-4-(trifluoromethyl)-3-pyridinesulfonamide).

The following sulfonylaminocarbonyltriazolinone herbicides illustratethe sulfonylaminocarbonyltriazolinones useful for this invention:flucarbazone(4,5-dihydro-3-methoxy-4-methyl-5-oxo-N-[[2-(trifluoromethoxy)phenyl]sulfonyl]-1H-1,2,4-triazole-1-carboxamide)and procarbazone (methyl2-[[[(4,5-dihydro-4-methyl-5-oxo-3-propoxy-1H-1,2,4-triazol-1-yl)carbonyl]amino]sulfonyl]benzoate).

Additional herbicides include phenmedipham, triazolinones, and theherbicides disclosed in WO2006/012981, herein incorporated by referencein its entirety.

The methods further comprise applying to the crop and the weeds in afield a sufficient amount of at least one herbicide to which the cropseeds or plants is tolerant, such as, for example, glyphosate, ahydroxyphenylpyruvatedioxygenase inhibitor (e.g., mesotrione orsulcotrione), a phytoene desaturase inhibitor (e.g., diflufenican), apigment synthesis inhibitor, sulfonamide, imidazolinone, bialaphos,phosphinothricin, azafenidin, butafenacil, sulfosate, glufosinate,triazolopyrimidine, pyrimidinyloxy(thio)benzoate, orsulonylaminocarbonyltriazolinone, an acetyl Co-A carboxylase inhibitorsuch as quizalofop-P-ethyl, a synthetic auxin such as quinclorac, or aprotox inhibitor to control the weeds without significantly damaging thecrop plants.

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. “Weed” as used herein refers to a plant which is notdesirable in a particular area. Conversely, a “crop plant” as usedherein refers to a plant which is desired in a particular area, such as,for example, a soybean plant. Thus, in some embodiments, a weed is anon-crop plant or a non-crop species, while in some embodiments, a weedis a crop species which is sought to be eliminated from a particulararea, such as, for example, an inferior and/or non-transgenic soybeanplant in a field planted with soybean event 3560.4.3.5, or a soybeanplant in a field planted with 3560.4.3.5. Weeds can be either classifiedinto two major groups: monocots and dicots.

Many plant species can be controlled (i.e., killed or damaged) by theherbicides described herein. Accordingly, the methods are useful incontrolling these plant species where they are undesirable (i.e., wherethey are weeds). These plant species include crop plants as well asspecies commonly considered weeds, including but not limited to speciessuch as: blackgrass (Alopecurus myosuroides), giant foxtail (Setariafaberi), large crabgrass (Digitaria sanguinalis), Surinam grass(Brachiaria decumbens), wild oat (Avena fatua), common cocklebur(Xanthium pensylvanicum), common lambsquarters (Chenopodium album),morning glory (Ipomoea coccinea), pigweed (Amaranthus spp.), velvetleaf(Abutilion theophrasti), common barnyardgrass (Echinochloa crus-galli),bermudagrass (Cynodon dactylon), downy brome (Bromus tectorum),goosegrass (Eleusine indica), green foxtail (Setaria viridis), Italianryegrass (Lolium multiflorum), Johnsongrass (Sorghum halepense), lessercanarygrass (Phalaris minor), windgrass (Apera spica-venti), woolycupgrass (Erichloa villosa), yellow nutsedge (Cyperus esculentus),common chickweed (Stellaria media), common ragweed (Ambrosiaartemisiifolia), Kochia scoparia, horseweed (Conyza canadensis), rigidryegrass (Lolium rigidum), goosegrass (Eleucine indica), hairy fleabane(Conyza bonariensis), buckhorn plantain (Plantago lanceolata), tropicalspiderwort (Commelina benghalensis), field bindweed (Convolvulusarvensis), purple nutsedge (Cyperus rotundus), redvine (Brunnichiaovata), hemp sesbania (Sesbania exaltata), sicklepod (Sennaobtusifolia), Texas blueweed (Helianthus ciliaris), and Devil's claws(Proboscidea louisianica). In other embodiments, the weed comprises aherbicide-resistant ryegrass, for example, a glyphosate resistantryegrass, a paraquat resistant ryegrass, a ACCase-inhibitor resistantryegrass, and a non-selective herbicide resistant ryegrass. In someembodiments, the undesired plants are proximate the crop plants.

As used herein, by “selectively controlled” it is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the cropplants are significantly damaged or killed.

In some embodiments, a soybean 3560.4.3.5 plant is not significantlydamaged by treatment with a particular herbicide applied to that plantat a dose equivalent to a rate of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 1.10, 120, 150, 170, 200, 300, 400, 500, 600,700, 800, 800, 1000, 2000, 3000, 4000, 5000 or more grams or ounces (1ounce=29.57 ml) of active ingredient or commercial product or herbicideformulation per acre or per hectare, whereas an appropriate controlplant is significantly damaged by the same treatment.

In specific embodiments, an effective amount of an ALS inhibitorherbicide comprises at least about 0.1, 1, 5, 10, 25, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 850, 900, 950,1000, 2000, 3000, 4000, 5000, or more grams or ounces (1 ounce=29.57 ml)of active ingredient per hectare. In other embodiments, an effectiveamount of an ALS inhibitor comprises at least about 0.1-50, about 25-75,about 50-100, about 100-110, about 110-120, about 120-130, about130-140, about 140-150, about 150-200, about 200-500, about 500-600,about 600-800, about 800-1000, or greater grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Any ALS inhibitor, for example,those listed in Table 1 can be applied at these levels.

In other embodiments, an effective amount of a sulfonylurea comprises atleast 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 5000 or more grams or ounces (1ounce=29.57 ml) of active ingredient per hectare. In other embodiments,an effective amount of a sulfonylurea comprises at least about 0.1-50,about 25-75, about 50-100, about 100-110, about 110-120, about 120-130,about 130-140, about 140-150, about 150-160, about 160-170, about170-180, about 190-200, about 200-250, about 250-300, about 300-350,about 350-400, about 400-450, about 450-500, about 500-550, about550-600, about 600-650, about 650-700, about 700-800, about 800-900,about 900-1000, about 1000-2000, or more grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Representative sulfonylureas thatcan be applied at this level are set forth in Table 1.

In other embodiments, an effective amount of asulfonylaminocarbonyltriazolinones, triazolopyrimidines,pyrimidinyloxy(thio)benzoates, and imidazolinones can comprise at leastabout 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1500, 1550, 1600, 1650, 1700, 1800,1850, 1900, 1950, 2000, 2500, 3500, 4000, 4500, 5000 or greater grams orounces (1 ounce=29.57 ml) active ingredient per hectare. In otherembodiments, an effective amount of a sulfonyluminocarbonyltriazolines,triazolopyrimidines, pyrimidinyloxy(thio)benzoates, or imidazolinonescomprises at least about 0.1-50, about 25-75, about 50-100, about100-110, about 110-120, about 120-130, about 130-140, about 140-150,about 150-160, about 160-170, about 170-180, about 190-200, about200-250, about 250-300, about 300-350, about 350-400, about 400-450,about 450-500, about 500-550, about 550-600, about 600-650, about650-700, about 700-800, about 800-900, about 900-1000, about 1000-2000,or more grams or ounces (1 ounce=29.57 ml) active ingredient perhectare.

Additional ranges of the effective amounts of herbicides can be found,for example, in various publications from University Extension services.See, for example, Bernards et al. (2006) Guide for Weed Management inNebraska (www.ianrpubs.url.edu/sendlt/ec130); Regher et al. (2005)Chemical Weed Control for Fields Crops, Pastures, Rangeland, andNoncropland, Kansas State University Agricultural Extension Station andCorporate Extension Service; Zollinger et al. (2006) North Dakota WeedControl Guide, North Dakota Extension Service, and the Iowa StateUniversity Extension at www.weeds.iastate.edu, each of which is hereinincorporated by reference.

In some embodiments, glyphosate is applied to an area of cultivationand/or to at least one plant in an area of cultivation at rates between8 and 32 ounces of acid equivalent per acre, or at rates between 10, 12,14, 16, 18, 20, 22, 24, 26, 28, and 30 ounces of acid equivalent peracre at the lower end of the range of application and between 12, 14,16, 18, 20, 22, 24, 26, 28, 30, and 32 ounces of acid equivalent peracre at the higher end of the range of application (1 ounce=29.57 ml).In other embodiments, glyphosate is applied at least at 1, 5, 10, 20,30, 40, 50, 60, 70, 80, 90 or greater ounce of active ingredient perhectare (1 ounce=29.57 ml). In some embodiments, a sulfonylureaherbicide is applied to a field and/or to at least one plant in a fieldat rates between 0.04 and 1.0 ounces of active ingredient per acre, orat rates between 0.1, 0.2, 0.4, 0.6, and 0.8 ounces of active ingredientper acre at the lower end of the range of application and between 0.2,0.4, 0.6, 0.8, and 1.0 ounces of active ingredient per acre at thehigher end of the range of application. (1 ounce=29.57 ml)

Glyphosate herbicides as a class contain the same active ingredient, butthe active ingredient is present as one of a number of different saltsand/or formulations. However, herbicides known to inhibit ALS vary intheir active ingredient as well as their chemical formulations. One ofskill in the art is familiar with the determination of the amount ofactive ingredient and/or acid equivalent present in a particular volumeand/or weight of herbicide preparation.

In some embodiments, an ALS inhibitor herbicide is employed. Rates atwhich the ALS inhibitor herbicide is applied to the crop, crop part,seed or area of cultivation can be any of the rates disclosed herein. Inspecific embodiments, the rate for the ALS inhibitor herbicide is about0.1 to about 5000 g ai/hectare, about 0.5 to about 300 g ai/hectare, orabout 1 to about 150 g ai/hectare.

Generally, a particular herbicide is applied to a particular field (andany plants growing in it) no more than 1, 2, 3, 4, 5, 6, 7, or 8 times ayear, or no more than 1, 2, 3, 4, or 5 times per growing season.

By “treated with a combination of” or “applying a combination of”herbicides to a crop, area of cultivation or field” it is intended thata particular field, crop or weed is treated with each of the herbicidesand/or chemicals indicated to be part of the combination so that adesired effect is achieved, i.e., so that weeds are selectivelycontrolled while the crop is not significantly damaged. In someembodiments, weeds which are susceptible to each of the herbicidesexhibit damage from treatment with each of the herbicides which isadditive or synergistic. The application of each herbicide and/orchemical may be simultaneous or the applications may be at differenttimes, so long as the desired effect is achieved. Furthermore, theapplication can occur prior to the planting of the crop.

The proportions of herbicides used with other herbicidal activeingredients in herbicidal compositions are generally in the ratio of5000:1 to 1:5000, 1000:1 to 1:1000, 100:1 to 1:100, 10:1 to 1:10 or 5:1to 1:5 by weight. The optimum ratios can be easily determined by thoseskilled in the art based on the weed control spectrum desired. Moreover,any combinations of ranges of the various herbicides disclosed in Table1 can also be applied in the methods.

Thus, in some embodiments, improved methods for selectively controllingweeds in a field are provided wherein the total herbicide applicationmay be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of that used in other methods.Similarly, in some embodiments, the amount of a particular herbicideused for selectively controlling weeds in a field may be less than 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, or 1% of the amount of that particular herbicide thatwould be used in other methods, i.e., methods not utilizing a plant ofthe invention.

In some embodiments, a 3560.4.3.5 soybean plant benefits from asynergistic effect wherein the herbicide tolerance conferred by theGLYAT polypeptide and the GM-HRA polypeptide is greater than expectedfrom simply combining the herbicide tolerance conferred by each geneseparately to a transgenic plant containing them individually. See,e.g., McCutchen et al. (1997) J. Econ. Entomol. 90: 1170-1180; Priesleret al. (1999) J. Econ. Entomol. 92: 598-603. As used herein, the terms“synergy,” “synergistic,” “synergistically” and derivations thereof,such as in a “synergistic effect” or a “synergistic herbicidecombination” or a “synergistic herbicide composition” refer tocircumstances under which the biological activity of a combination ofherbicides, such as at least a first herbicide and a second herbicide,is greater than the sum of the biological activities of the individualherbicides. Synergy, expressed in terms of a “Synergy Index (SI),”generally can be determined by the method described by Kull et al.Applied Microbiology 9, 538 (1961). See also Colby “CalculatingSynergistic and Antagonistic Responses of Herbicide Combinations,” Weeds15, 20-22 (1967).

In other instances, the herbicide tolerance conferred on a 3560.4.3.5plant is additive; that is, the herbicide tolerance profile conferred bythe herbicide tolerance genes is what would be expected from simplycombining the herbicide tolerance conferred by each gene separately to atransgenic plant containing them individually. Additive and/orsynergistic activity for two or more herbicides against key weed specieswill increase the overall effectiveness and/or reduce the actual amountof active ingredient(s) needed to control said weeds. Where such synergyis observed, the plant may display tolerance to a higher dose or rate ofherbicide and/or the plant may display tolerance to additionalherbicides or other chemicals beyond those to which it would be expectedto display tolerance. For example, a 3560.4.3.5 soybean plant may showtolerance to organophosphate compounds such as insecticides and/orinhibitors of 4-hydroxyphenylpyruvate dioxygenase.

Thus, for example, the 3560.4.3.5 soybean plants can exhibit greaterthan expected tolerance to various herbicides, including but not limitedto glyphosate, ALS inhibitor chemistries, and sulfonylurea herbicides.The 3560.4.3.5 soybean plants may show tolerance to a particularherbicide or herbicide combination that is at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,300%, 400%, or 500% or more higher than the tolerance of an appropriatecontrol plant that contains only a single herbicide tolerance gene whichconfers tolerance to the same herbicide or herbicide combination. Thus,3560.4.3.5 soybean plants may show decreased damage from the same doseof herbicide in comparison to an appropriate control plant, or they mayshow the same degree of damage in response to a much higher dose ofherbicide than the control plant. Accordingly, in specific embodiments,a particular herbicide used for selectively containing weeds in a fieldis more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80%, 90%, 100% or greater than the amount of that particularherbicide that would be used in other methods, i.e., methods notutilizing a plant of the invention.

In the same manner, in some embodiments, a 3560.4.3.5 soybean plantshows improved tolerance to a particular formulation of a herbicideactive ingredient in comparison to an appropriate control plant.Herbicides are sold commercially as formulations which typically includeother ingredients in addition to the herbicide active ingredient; theseingredients are often intended to enhance the efficacy of the activeingredient. Such other ingredients can include, for example, safenersand adjuvants (see, e.g., Green and Foy (2003) “Adjuvants: Tools forEnhancing Herbicide Performance,” in Weed Biology and Management, ed.Inderjit (Kluwer Academic Publishers, The Netherlands)). Thus, a3560.4.3.5 soybean plant can show tolerance to a particular formulationof a herbicide (e.g., a particular commercially available herbicideproduct) that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%,15%, 17%, 20%, 22%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%, 600%,700%, 800%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%, 1500%, 1600%,1700%, 1800%, 1900%, or 2000% or more higher than the tolerance of anappropriate control plant that contains only a single herbicidetolerance gene which confers tolerance to the same herbicideformulation.

In some embodiments, a 3560.4.3.5 soybean plant shows improved toleranceto a herbicide or herbicide class to which at least one other herbicidetolerance gene confers tolerance as well as improved tolerance to atleast one other herbicide or chemical which has a different mechanism orbasis of action than either glyphosate or the herbicide corresponding tosaid at least one other herbicide tolerance gene. This surprisingbenefit finds use in methods of growing crops that comprise treatmentwith various combinations of chemicals, including, for example, otherchemicals used for growing crops. Thus, for example, a 3560.4.3.5soybean plant may also show improved tolerance to chlorpyrifos, asystemic organophosphate insecticide. Thus, further provided is a3560.4.3.5 soybean plant that confers tolerance to glyphosate (i.e., aglyat gene) and a sulfonylurea herbicide tolerance gene which showsimproved tolerance to chemicals which affect the cytochrome P450 gene,and methods of use thereof. In some embodiments, the 3560.4.3.5 soybeanplants also show improved tolerance to dicamba. In these embodiments,the improved tolerance to dicamba may be evident in the presence ofglyphosate and a sulfonylurea herbicide.

In other methods, a herbicide combination is applied over a 3560.4.3.5soybean plant, where the herbicide combination produces either anadditive or a synergistic effect for controlling weeds. Suchcombinations of herbicides can allow the application rate to be reduced,a broader spectrum of undesired vegetation to be controlled, improvedcontrol of the undesired vegetation with fewer applications, more rapidonset of the herbicidal activity, or more prolonged herbicidal activity.

An “additive herbicidal composition” has a herbicidal activity that isabout equal to the observed activities of the individual components. A“synergistic herbicidal combination” has a herbicidal activity higherthan what can be expected based on the observed activities of theindividual components when used alone. Accordingly, the presentlydisclosed subject matter provides a synergistic herbicide combination,wherein the degree of weed control of the mixture exceeds the sum ofcontrol of the individual herbicides. In some embodiments, the degree ofweed control of the mixture exceeds the sum of control of the individualherbicides by any statistically significant amount including, forexample, about 1% to 5%, about 5% to about 10%, about 10% to about 20%,about 20% to about 30%, about 30% to 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, about 90% to about 100%, about 100% to 120% orgreater. Further, a “synergistically effective amount” of a herbiciderefers to the amount of one herbicide necessary to elicit a synergisticeffect in another herbicide present in the herbicide composition. Thus,the term “synergist,” and derivations thereof, refer to a substance thatenhances the activity of an active ingredient (ai), i.e., a substance ina formulation from which a biological effect is obtained, for example, aherbicide.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for controlling weeds in an area of cultivation. Insome embodiments, the method comprises: (a) planting the area with a3560.4.3.5 crop seeds or crop plants; and (b) applying to the weed, thecrop plants, a crop part, the area of cultivation, or a combinationthereof, an effective amount of a herbicide composition comprising atleast one of a synergistically effective amount of glyphosate and asynergistically effective amount of an ALS inhibitor (for example, butnot limited to, a sulfonylurea herbicide), or agriculturally suitablesalts thereof, wherein at least one of: (i) the synergisticallyeffective amount of the glyphosate is lower than an amount of glyphosaterequired to control the weeds in the absence of the sulfonylureaherbicide; (ii) the synergistically effective amount of the ALSinhibitor herbicide is lower than an amount of the ALS inhibitorrequired to control the weeds in the absence of glyphosate; and (iii)combinations thereof; and wherein the effective amount of the herbicidecomposition is tolerated by the crop seeds or crop plants and controlsthe weeds in the area of cultivation.

In some embodiments, the herbicide composition used in the presentlydisclosed method for controlling weeds comprises a synergisticallyeffective amount of glyphosate and a sulfonylurea herbicide. In furtherembodiments, the presently disclosed synergistic herbicide compositioncomprises glyphosate and a sulfonylurea herbicide selected from thegroup consisting of metsulfuron-methyl, chlorsulfuron, and triasulfuron.

In particular embodiments, the synergistic herbicide combination furthercomprises an adjuvant such as, for example, an ammonium sulfate-basedadjuvant, e.g., ADD-UP® (Wenkem S. A., Halfway House, Midrand, SouthAfrica). In additional embodiments, the presently disclosed synergisticherbicide compositions comprise an additional herbicide, for example, aneffective amount of a pyrimidinyloxy(thio)benzoate herbicide. In someembodiments, the pyrimidinyloxy(thio)benzoate herbicide comprisesbispyribac, e.g., (VELOCITY®, Valent U.S.A. Corp., Walnut Creek, Calif.,United States of America), or an agriculturally suitable salt thereof.

In some embodiments of the presently disclosed method for controllingundesired plants, the glyphosate is applied pre-emergence,post-emergence or pre- and post-emergence to the undesired plants orplant crops; and/or the ALS inhibitor herbicide (i.e., the sulfonylureaherbicide) is applied pre-emergence, post-emergence or pre- andpost-emergence to the undesired plants or plant crops. In otherembodiments, the glyphosate and/or the ALS inhibitor herbicide (i.e.,the sulfonylurea herbicide) are applied together or are appliedseparately. In yet other embodiments, the synergistic herbicidecomposition is applied, e.g. step (b) above, at least once prior toplanting the crop(s) of interest, e.g., step (a) above.

Weeds that can be difficult to control with glyphosate alone in fieldswhere a crop is grown (such as, for example, a soybean crop) include butare not limited to the following: horseweed (e.g., Conyza canadensis);rigid ryegrass (e.g., Lolium rigidum); goosegrass (e.g., Eleusineindica); Italian ryegrass (e.g., Lolium multiflorum); hairy fleabane(e.g., Conyza bonariensis); buckhorn plantain (e.g., Plantagolanceolata); common ragweed (e.g., Ambrosia artemisifolia); morningglory (e.g., Ipomoea spp.); waterhemp (e.g., Amaranthus spp.); fieldbindweed (e.g., Convolvulus arvensis); yellow nutsedge (e.g., Cyperusesculentus); common lambsquarters (e.g., Chenopodium album); wildbuckwheat (e.g., Polygonium convolvulus); velvetleaf (e.g., Abutilontheophrasti); kochia (e.g., Kochia scoparia); and Asiatic dayflower(e.g., Commelina spp.). In areas where such weeds are found, the3560.4.3.5 soybeans are particularly useful in allowing the treatment ofa field (and therefore any crop growing in the field) with combinationsof herbicides that would cause unacceptable damage to crop plants thatdid not contain both of these polynucleotides. Plants that are tolerantto glyphosate and other herbicides such as, for example, sulfonylurea,imidazolinone, triazolopyrimidine, pyrimidinyl(thio)benzoate, and/orsulfonylaminocarbonyltriazolinone herbicides in addition to beingtolerant to at least one other herbicide with a different mode of actionor site of action are particularly useful in situations where weeds aretolerant to at least two of the same herbicides to which the plants aretolerant. In this manner, plants of the invention make possible improvedcontrol of weeds that are tolerant to more than one herbicide.

For example, some commonly used treatments for weed control in fieldswhere current commercial crops (including, for example, soybeans) aregrown include glyphosate and, optionally, 2,4-D; this combination,however, has some disadvantages. Particularly, there are weed speciesthat it does not control well and it also does not work well for weedcontrol in cold weather. Another commonly used treatment for weedcontrol in soybean fields is the sulfonylurea herbicidechlorimuron-ethyl, which has significant residual activity in the soiland thus maintains selective pressure on all later-emerging weedspecies, creating a favorable environment for the growth and spread ofsulfonylurea-resistant weeds. However, the 3560.4.3.5 soybean can betreated with herbicides (e.g., chlorimuron-ethyl) and combinations ofherbicides that would cause unacceptable damage to standard plantvarieties. Thus, for example, fields containing the 3560.4.3.5 soybeancan be treated with sulfonylurea, imidazolinone, triazolopyrimidines,pyrimidiny(thio)benzoates, and/or sulfonylaminocarbonyltriazonlinonesuch as the sulfonylurea chlorimuron-ethyl, either alone or incombination with other herbicides. For example, fields containingsoybean plants of the invention can be treated with a combination ofglyphosate and tribenuron-methyl (available commercially as Express®).This combination has several advantages for weed control under somecircumstances, including the use of herbicides with different modes ofaction and the use of herbicides having a relatively short period ofresidual activity in the soil. A herbicide having a relatively shortperiod of residual activity is desirable, for example, in situationswhere it is important to reduce selective pressure that would favor thegrowth of herbicide-tolerant weeds. Of course, in any particularsituation where weed control is required, other considerations may bemore important, such as, for example, the need to prevent thedevelopment of and/or appearance of weeds in a field prior to planting acrop by using a herbicide with a relatively long period of residualactivity. The 3560.4.3.5 soybean plants can also be treated withherbicide combinations that include at least one of nicosulfuron,metsulfuron-methyl, tribenuron-methyl, thifensulfuron-methyl, and/orrimsulfuron. Treatments that include both tribenuron-methyl andthifensulfuron-methyl may be particularly useful.

Other commonly used treatments for weed control in fields where currentcommercial varieties of crops (including, for example, soybeans) aregrown include the sulfonylurea herbicide thifensulfuron-methyl(available commercially as Harmony GTE). However, one disadvantage ofthifensulfuron-methyl is that the higher application rates required forconsistent weed control often cause injury to a crop growing in the samefield. The 3560.4.3.5 soybean plants can be treated with a combinationof glyphosate and thifensulfuron-methyl, which has the advantage ofusing herbicides with different modes of action. Thus, weeds that areresistant to either herbicide alone are controlled by the combination ofthe two herbicides, and the 3560.4.3.5 soybean plants are notsignificantly damaged by the treatment.

Other herbicides which are used for weed control in fields where currentcommercial varieties of crops (including, for example, soybeans) aregrown are the triazolopyrimidine herbicide cloransulam-methyl (availablecommercially as FirstRate®) and the imidazolinone herbicide imazaquin(available commercially as Sceptor®). When these herbicides are usedindividually they may provide only marginal control of weeds. However,fields containing the 3560.4.3.5 soybean can be treated, for example,with a combination of glyphosate (e.g., Roundup® (glyphosateisopropylamine salt)), imazapyr (currently available commercially asArsenal®), chlorimuron-ethyl (currently available commercially asClassic®), quizalofop-P-ethyl (currently available commercially asAssure II®), and fomesafen (currently available commercially asFlexstar®). This combination has the advantage of using herbicides withdifferent modes of action. Thus, weeds that are tolerant to just one orseveral of these herbicides are controlled by the combination of thefive herbicides, and the 3560.4.3.5 soybeans are not significantlydamaged by treatment with this herbicide combination. This combinationprovides an extremely broad spectrum of protection against the type ofherbicide-tolerant weeds that might be expected to arise and spreadunder current weed control practices.

Fields containing the 3560.4.3.5 soybean plants may also be treated, forexample, with a combination of herbicides including glyphosate,rimsulfuron, and dicamba or mesotrione. This combination may beparticularly useful in controlling weeds which have developed sometolerance to herbicides which inhibit ALS. Another combination ofherbicides which may be particularly useful for weed control includesglyphosate and at least one of the following: metsulfuron-methyl(commercially available as Ally®), imazapyr (commercially available asArsenal®), imazethapyr, imazaquin, and sulfentrazone. It is understoodthat any of the combinations discussed above or elsewhere herein mayalso be used to treat areas in combination with any other herbicide oragricultural chemical.

Some commonly-used treatments for weed control in fields where currentcommercial crops (including, for example, maize) are grown includeglyphosate (currently available commercially as Roundup®), rimsulfuron(currently available commercially as Resolve® or Matrix®), dicamba(commercially available as Clarity®), atrazine, and mesotrione(commercially available as Callisto®). These herbicides are sometimesused individually due to poor crop tolerance to multiple herbicides.Unfortunately, when used individually, each of these herbicides hassignificant disadvantages. Particularly, the incidence of weeds that aretolerant to individual herbicides continues to increase, renderingglyphosate less effective than desired in some situations. Rimsulfuronprovides better weed control at high doses which can cause injury to acrop, and alternatives such as dicamba are often more expensive thanother commonly-used herbicides. However, 3560.4.3.5 soybean can betreated with herbicides and combinations of herbicides that would causeunacceptable damage to standard plant varieties, including combinationsof herbicides that comprise rimsulfuron and/or dicamba. Other suitablecombinations of herbicides for use with 3560.4.3.5 soybean plantsinclude glyphosate, sulfonylurea, imidazolinone, triazolopyrimidine,pryimidinyloxy(thio)benzoates, and/or sulfonylaminocarbonyltriazonlinoneherbicides, including, for example, and at least one of the following:metsulfuron-methyl, tribenuron-methyl, chlorimuron-ethyl, imazethapyr,imazapyr, and imazaquin.

For example, 3560.4.3.5 soybean plants can be treated with a combinationof glyphosate and rimsulfuron, or a combination or rimsulfuron and atleast one other herbicide. 3560.4.3.5 soybean plants can also be treatedwith a combination of glyphosate, rimsulfuron, and dicamba, or acombination of glyphosate, rimsulfuron, and at least one otherherbicide. In some embodiments, at least one other herbicide has adifferent mode of action than both glyphosate and rimsulfuron. Thecombination of glyphosate, rimsulfuron, and dicamba has the advantagethat these herbicides have different modes of action and short residualactivity, which decreases the risk of incidence and spread ofherbicide-tolerant weeds.

Some commonly-used treatments for weed control in fields where currentcommercial crops are grown include glyphosate (currently availablecommercially as Roundup®), chlorimuron-ethyl, tribenuron-methyl,rimsulfuron (currently available commercially as Resolve® or Matrix®),imazethapyr, imazapyr, and imazaquin. Unfortunately, when usedindividually, each of these herbicides has significant disadvantages.Particularly, the incidence of weeds that are tolerant to individualherbicides continues to increase, rendering each individual herbicideless effective than desired in some situations. However, 3560.4.3.5soybean can be treated with a combination of herbicides that would causeunacceptable damage to standard plant varieties, including combinationsof herbicides that include at least one of those mentioned above.

In the methods, a herbicide may be formulated and applied to an area ofinterest such as, for example, a field or area of cultivation, in anysuitable manner. A herbicide may be applied to a field in any form, suchas, for example, in a liquid spray or as solid powder or granules. Inspecific embodiments, the herbicide or combination of herbicides thatare employed in the methods comprises a tankmix or a premix. A herbicidemay also be formulated, for example, as a “homogenous granule blend”produced using blends technology (see, e.g., U.S. Pat. No. 6,022,552,entitled “Uniform Mixtures of Pesticide Granules”). The blendstechnology of U.S. Pat. No. 6,022,552 produces a nonsegregating blend(i.e., a “homogenous granule blend”) of formulated crop protectionchemicals in a dry granule form that enables delivery of customizedmixtures designed to solve specific problems. A homogenous granule blendcan be shipped, handled, subsampled, and applied in the same manner astraditional premix products where multiple active ingredients areformulated into the same granule.

Briefly, a “homogenous granule blend” is prepared by mixing together atleast two extruded formulated granule products. In some embodiments,each granule product comprises a registered formulation containing asingle active ingredient which is, for example, a herbicide, afungicide, and/or an insecticide. The uniformity (homogeneity) of a“homogenous granule blend” can be optimized by controlling the relativesizes and size distributions of the granules used in the blend. Thediameter of extruded granules is controlled by the size of the holes inthe extruder die, and a centrifugal sifting process may be used toobtain a population of extruded granules with a desired lengthdistribution (see, e.g., U.S. Pat. No. 6,270,025).

A homogenous granule blend is considered to be “homogenous” when it canbe subsampled into appropriately sized aliquots and the composition ofeach aliquot will meet the required assay specifications. To demonstratehomogeneity, a large sample of the homogenous granule blend is preparedand is then subsampled into aliquots of greater than the minimumstatistical sample size.

In non-limiting embodiments, the 3560.4.5.3 soybean plant can be treatedwith herbicides (e.g., chlorimuron-ethyl and combinations of otherherbicides that without the 3560.4.3.5 event would have causedunacceptable crop response to plant varieties without the glyphosate/ALSinhibitor genetics). Thus, for example, fields planted with andcontaining 3560.4.3.5 soybeans can be treated with sulfonylurea,imidazolinone, triazolopyrimidine, pyrimidinyl(thio)benzoate, and/orsulfonylaminocarbonyltriazonlinone herbicides, either alone or incombination with other herbicides. Since ALS inhibitor chemistries havedifferent herbicidal attributes, blends of ALS inhibitor plus otherchemistries will provide superior weed management strategies includingvarying and increased weed spectrum, the ability to provide specifiedresidual activity (SU/ALS inhibitor chemistry with residual activityleads to improved foliar activity which leads to a wider window betweenglyphosate applications, as well as, an added period of control ifweather conditions prohibit timely application).

Blends also afford the ability to add other agrochemicals at normal,labeled use rates such as additional herbicides (a 3^(rd)/4^(th)mechanism of action), fungicides, insecticides, plant growth regulatorsand the like thereby saving costs associated with additionalapplications.

Any herbicide formulation applied over the 3560.4.3.5 soybean plant canbe prepared as a “tank-mix” composition. In such embodiments, eachingredient or a combination of ingredients can be stored separately fromone another. The ingredients can then be mixed with one another prior toapplication. Typically, such mixing occurs shortly before application.In a tank-mix process, each ingredient, before mixing, typically ispresent in water or a suitable organic solvent. For additional guidanceregarding the art of formulation, see T. S. Woods, “The Formulator'sToolbox—Product Forms for Modern Agriculture” Pesticide Chemistry andBioscience, The Food-Environment Challenge, T. Brooks and T. R. Roberts,Eds., Proceedings of the 9th International Congress on PesticideChemistry, The Royal Society of Chemistry, Cambridge, 1999, pp. 120-133.See also U.S. Pat. No. 3,235,361, Col. 6, line 16 through Col. 7, line19 and Examples 10-41; U.S. Pat. No. 3,309,192, Col. 5, line 43 throughCol. 7, line 62 and Examples 8, 12, 15, 39, 41, 52, 53, 58, 132,138-140, 162-164, 166, 167 and 169-182; U.S. Pat. No. 2,891,855, Col. 3,line 66 through Col. 5, line 17 and Examples 1-4; Klingman, Weed Controlas a Science, John Wiley and Sons, Inc., New York, 1961, pp 81-96; andHance et al., Weed Control Handbook, 8th Ed., Blackwell ScientificPublications, Oxford, 1989, each of which is incorporated herein byreference in their entirety.

The methods further allow for the development of herbicide combinationsto be used with the 3560.4.3.5 soybean plants. In such methods, theenvironmental conditions in an area of cultivation are evaluated.Environmental conditions that can be evaluated include, but are notlimited to, ground and surface water pollution concerns, intended use ofthe crop, crop tolerance, soil residuals, weeds present in area ofcultivation, soil texture, pH of soil, amount of organic matter in soil,application equipment, and tillage practices. Upon the evaluation of theenvironmental conditions, an effective amount of a combination ofherbicides can be applied to the crop, crop part, seed of the crop orarea of cultivation.

In some embodiments, the herbicide applied to the 3560.4.3.5 soybeanplants serves to prevent the initiation of growth of susceptible weedsand/or serve to cause damage to weeds that are growing in the area ofinterest. In some embodiments, the herbicide or herbicide mixture exertthese effects on weeds affecting crops that are subsequently planted inthe area of interest (i.e., field or area of cultivation). In themethods, the application of the herbicide combination need not occur atthe same time. So long as the field in which the crop is plantedcontains detectable amounts of the first herbicide and the secondherbicide is applied at some time during the period in which the crop isin the area of cultivation, the crop is considered to have been treatedwith a mixture of herbicides according to the invention. Thus, methodsencompass applications of herbicide which are “preemergent,” “at plant,“postemergent,” “preplant incorporation” and/or seed treatment prior toplanting.

In one embodiment, methods are provided for coating seeds. The methodscomprise coating a seed with an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein). The seeds canthen be planted in an area of cultivation. Further provided are seedshaving a coating comprising an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein).

“Preemergent” refers to a herbicide which is applied to an area ofinterest (e.g., a field or area of cultivation) before a plant emergesvisibly from the soil. “Postemergent” refers to a herbicide which isapplied to an area after a plant emerges visibly from the soil. In someinstances, the terms “preemergent” and “postemergent” are used withreference to a weed in an area of interest, and in some instances theseterms are used with reference to a crop plant in an area of interest.When used with reference to a weed, these terms may apply to only aparticular type of weed or species of weed that is present or believedto be present in the area of interest. While any herbicide may beapplied in a preemergent and/or postemergent treatment, some herbicidesare known to be more effective in controlling a weed or weeds whenapplied either preemergence or postemergence. For example, rimsulfuronhas both preemergence and postemergence activity, while other herbicideshave predominately preemergence (metolachlor) or postemergence(glyphosate) activity. These properties of particular herbicides areknown in the art and are readily determined by one of skill in the art.Further, one of skill in the art would readily be able to selectappropriate herbicides and application times for use with the transgenicplants of the invention and/or on areas in which transgenic plants ofthe invention are to be planted. “Preplant incorporation” involves theincorporation of compounds into the soil prior to planting.

Thus, improved methods of growing a crop and/or controlling weeds areprovided such as, for example, “pre-planting burn down,” wherein an areais treated with herbicides prior to planting the crop of interest inorder to better control weeds. Further provided are methods of growing acrop and/or controlling weeds which are “no-till” or “low-till” (alsoreferred to as “reduced tillage”). In such methods, the soil is notcultivated or is cultivated less frequently during the growing cycle incomparison to traditional methods; these methods can save costs thatwould otherwise be incurred due to additional cultivation, includinglabor and fuel costs.

The methods encompass the use of simultaneous and/or sequentialapplications of multiple classes of herbicides. In some embodiments, themethods involve treating a plant of the invention and/or an area ofinterest (e.g., a field or area of cultivation) and/or weed with justone herbicide or other chemical such as, for example, a sulfonylureaherbicide.

The time at which a herbicide is applied to an area of interest (and anyplants therein) may be important in optimizing weed control. The time atwhich a herbicide is applied may be determined with reference to thesize of plants and/or the stage of growth and/or development of plantsin the area of interest, e.g., crop plants or weeds growing in the area.The stages of growth and/or development of plants are known in the art.For example, soybean plants normally progress through vegetative growthstages known as V_(E) (emergence), V_(C) (unifoliolate), V₁ (firsttrifoliolate), and V₂ to V_(N). Soybeans then switch to the reproductivegrowth phase in response to photoperiod cues; reproductive stagesinclude R₁ (beginning bloom), R₂ (full bloom), R₃ (beginning pod), R₄(full pod), R₅ (beginning seed), R₆ (full seed), R₇ (beginningmaturity), and R₈ (full maturity). Thus, for example, the time at whicha herbicide or other chemical is applied to an area of interest in whichplants are growing may be the time at which some or all of the plants ina particular area have reached at least a particular size and/or stageof growth and/or development, or the time at which some or all of theplants in a particular area have not yet reached a particular sizeand/or stage of growth and/or development.

In some embodiments, the 3560.4.3.5 soybean plants show improvedtolerance to postemergence herbicide treatments. For example, the3560.4.3.5 plants may be tolerant to higher doses of herbicide, tolerantto a broader range of herbicides (i.e., tolerance to more ALS inhibitorchemistries), and/or may be tolerant to doses of herbicide applied atearlier or later times of development in comparison to an appropriatecontrol plant. For example, in some embodiments, the 3560.4.3.5 soybeanplants show an increased resistance to morphological defects that areknown to result from treatment at particular stages of development.

Different chemicals such as herbicides have different “residual”effects, i.e., different amounts of time for which treatment with thechemical or herbicide continues to have an effect on plants growing inthe treated area. Such effects may be desirable or undesirable,depending on the desired future purpose of the treated area (e.g., fieldor area of cultivation). Thus, a crop rotation scheme may be chosenbased on residual effects from treatments that will be used for eachcrop and their effect on the crop that will subsequently be grown in thesame area. One of skill in the art is familiar with techniques that canbe used to evaluate the residual effect of a herbicide; for example,generally, glyphosate has very little or no soil residual activity,while herbicides that act to inhibit ALS vary in their residual activitylevels. Residual activities for various herbicides are known in the art,and are also known to vary with various environmental factors such as,for example, soil moisture levels, temperature, pH, and soil composition(texture and organic matter). The 3560.4.3.5 soybean plants findparticular use in methods of growing a crop where improved tolerance toresidual activity of a herbicide is beneficial.

For example, in one embodiment, the 3560.4.3.5 soybean plants have animproved tolerance to glyphosate as well as to ALS inhibitor chemistries(such as sulfonylurea herbicides) when applied individually, and furtherprovide improved tolerance to combinations of herbicides such asglyphosate and/or ALS inhibitor chemistries. Moreover, the transgenicplants disclosed herein provide improved tolerance to treatment withadditional chemicals commonly used on crops in conjunction withherbicide treatments, such as safeners, adjuvants such as ammoniumsulfonate and crop oil concentrate, and the like.

The term “safener” refers to a substance that when added to a herbicideformulation eliminates or reduces the phytotoxic effects of theherbicide to certain crops. One of ordinary skill in the art wouldappreciate that the choice of safener depends, in part, on the cropplant of interest and the particular herbicide or combination ofherbicides included in the synergistic herbicide composition. Exemplarysafeners suitable for use with the presently disclosed herbicidecompositions include, but are not limited to, those disclosed in U.S.Pat. Nos. 4,808,208; 5,502,025; 6,124,240 and U.S. Patent ApplicationPublication Nos. 2006/0148647; 2006/0030485; 2005/0233904; 2005/0049145;2004/0224849; 2004/0224848; 2004/0224844; 2004/0157737; 2004/0018940;2003/0171220; 2003/0130120; 2003/0078167, the disclosures of which areincorporated herein by reference in their entirety. The methods caninvolve the use of herbicides in combination with herbicide safenerssuch as benoxacor, BCS (1-bromo-4-[(chloromethyl) sulfonyl]benzene),cloquintocet-mexyl, cyometrinil, dichlormid,2-(dichloromethyl)-2-methyl-1,3-dioxolane (MG 191), fenchlorazole-ethyl,fenclorim, flurazole, fluxofenim, furilazole, isoxadifen-ethyl,mefenpyr-diethyl, methoxyphenone((4-methoxy-3-methylphenyl)(3-methylphenyl)-methanone), naphthalicanhydride (1,8-naphthalic anhydride) and oxabetrinil to increase cropsafety. Antidotally effective amounts of the herbicide safeners can beapplied at the same time as the compounds, or applied as seedtreatments. Therefore an aspect of the present invention relates to theuse of a mixture comprising glyphosate, at least one other herbicide,and an antidotally effective amount of a herbicide safener.

Seed treatment is particularly useful for selective weed control,because it physically restricts antidoting to the crop plants. Thereforea particularly useful embodiment is a method for selectively controllingthe growth of weeds in a field comprising treating the seed from whichthe crop is grown with an antidotally effective amount of safener andtreating the field with an effective amount of herbicide to controlweeds. Antidotally effective amounts of safeners can be easilydetermined by one skilled in the art through simple experimentation. Anantidotally effective amount of a safener is present where a desiredplant is treated with the safener so that the effect of a herbicide onthe plant is decreased in comparison to the effect of the herbicide on aplant that was not treated with the safener; generally, an antidotallyeffective amount of safener prevents damage or severe damage to theplant treated with the safener. One of skill in the art is capable ofdetermining whether the use of a safener is appropriate and determiningthe dose at which a safener should be administered to a crop.

In specific embodiments, the herbicide or herbicide combination appliedto the 3560.4.3.5 plant acts as a safener. In this embodiment, a firstherbicide or a herbicide mixture is applied at an antidotally effectamount to the plant. Accordingly, a method for controlling weeds in anarea of cultivation is provided. The method comprises planting the areawith crop seeds or plants which comprise a first polynucleotide encodinga polypeptide that can confer tolerance to glyphosate operably linked toa promoter active in a plant; and, a second polynucleotide encoding anALS inhibitor-tolerant polypeptide operably linked to a promoter activein a plant. A combination of herbicides comprising at least an effectiveamount of a first and a second herbicide is applied to the crop, croppart, weed or area of cultivation thereof. The effective amount of theherbicide combination controls weeds; and, the effective amount of thefirst herbicide is not tolerated by the crop when applied alone whencompared to a control crop that has not been exposed to the firstherbicide; and, the effective amount of the second herbicide issufficient to produce a safening effect, wherein the safening effectprovides an increase in crop tolerance upon the application of the firstand the second herbicide when compared to the crop tolerance when thefirst herbicide is applied alone.

In specific embodiments, the combination of safening herbicidescomprises a first ALS inhibitor and a second ALS inhibitor. In otherembodiments, the safening effect is achieved by applying an effectiveamount of a combination of glyphosate and at least one ALS inhibitorchemistry. In still other embodiments, a safening affect is achievedwhen the 3560.4.3.5 soybean crops, crop part, crop seed, weed, or areaof cultivation is treated with at least one herbicide from thesulfonylurea family of chemistries in combination with at least oneherbicide from the ALS family of chemistries (such as, for example, animidazolinone).

Such mixtures provide increased crop tolerance (i.e., a decrease inherbicidal injury). This method allows for increased application ratesof the chemistries post or pre-treatment. Such methods find use forincreased control of unwanted or undesired vegetation. In still otherembodiments, a safening affect is achieved when the 3560.4.3.5 soybeancrops, crop part, crop seed, weed, or area of cultivation is treatedwith at least one herbicide from the sulfonylurea family of chemistry incombination with at least one herbicide from the imidazolinone family.This method provides increased crop tolerance (i.e., a decrease inherbicidal injury). In specific embodiments, the sulfonylurea comprisesrimsulfuron and the imidazolinone comprises imazethapyr. In otherembodiments, glyphosate is also applied to the crop, crop part, or areaof cultivation.

As used herein, an “adjuvant” is any material added to a spray solutionor formulation to modify the action of an agricultural chemical or thephysical properties of the spray solution. See, for example, Green andFoy (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” inWeed Biology and Management, ed. Inderjit (Kluwer Academic Publishers,The Netherlands). Adjuvants can be categorized or subclassified asactivators, acidifiers, buffers, additives, adherents, antiflocculants,antifoamers, defoamers, antifreezes, attractants, basic blends,chelating agents, cleaners, colorants or dyes, compatibility agents,cosolvents, couplers, crop oil concentrates, deposition agents,detergents, dispersants, drift control agents, emulsifiers, evaporationreducers, extenders, fertilizers, foam markers, formulants, inerts,humectants, methylated seed oils, high load COCs, polymers, modifiedvegetable oils, penetrators, repellants, petroleum oil concentrates,preservatives, rainfast agents, retention aids, solubilizers,surfactants, spreaders, stickers, spreader stickers, synergists,thickeners, translocation aids, uv protectants, vegetable oils, waterconditioners, and wetting agents.

In addition, methods can comprise the use of a herbicide or a mixture ofherbicides, as well as, one or more other insecticides, fungicides,nematocides, bactericides, acaricides, growth regulators,chemosterilants, semiochemicals, repellents, attractants, pheromones,feeding stimulants or other biologically active compounds orentomopathogenic bacteria, virus, or fungi to form a multi-componentmixture giving an even broader spectrum of agricultural protection.Examples of such agricultural protectants which can be used in methodsinclude: insecticides such as abamectin, acephate, acetamiprid,amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl,bifenthrin, bifenazate, buprofezin, carbofuran, cartap, chlorfenapyr,chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide,clothianidin, cyflumetofen, cyfluthrin, beta-cyfluthrin, cyhalothrin,lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, dieldrin, diflubenzuron, dimefluthrin,dimethoate, dinotefuran, diofenolan, emamectin, endosulfan,esfenvalerate, ethiprole, fenothiocarb, fenoxycarb, fenpropathrin,fenvalerate, fipronil, flonicamid, flubendiamide, flucythrinate,tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos,halofenozide, hexaflumuron, hydramethylnon, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaflumizone, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,metofluthrin, monocrotophos, methoxyfenozide, nitenpyram, nithiazine,novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl,permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb,profenofos, profluthrin, pymetrozine, pyrafluprole, pyrethrin,pyridalyl, pyriprole, pyriproxyfen, rotenone, ryanodine, spinosad,spirodiclofen, spiromesifen (BSN 2060), spirotetramat, sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,triazamate, trichlorfon and triflumuron; fungicides such as acibenzolar,aldimorph, amisulbrom, azaconazole, azoxystrobin, benalaxyl, benomyl,benthiavalicarb, benthiavalicarb-isopropyl, binomial, biphenyl,bitertanol, blasticidin-S, Bordeaux mixture (Tribasic copper sulfate),boscalid/nicobifen, bromuconazole, bupirimate, buthiobate, carboxin,carpropamid, captafol, captan, carbendazim, chloroneb, chlorothalonil,chlozolinate, clotrimazole, copper oxychloride, copper salts such ascopper sulfate and copper hydroxide, cyazofamid, cyflunamid, cymoxanil,cyproconazole, cyprodinil, dichlorfluanid, diclocymet, diclomezine,dicloran, diethofencarb, difenoconazole, dimethomorph, dimoxystrobin,diniconazole, diniconazole-M, dinocap, discostrobin, dithianon,dodemorph, dodine, econazole, etaconazole, edifenphos, epoxiconazole,ethaboxam, ethirimol, etridiazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid, fenfuram, fenhexamid, fenoxanil, fenpiclonil,fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, ferbam,ferfurazoate, ferimzone, fluazinam, fludioxonil, flumetover,fluopicolide, fluoxastrobin, fluquinconazole, fluquinconazole,flusilazole, flusulfamide, flutolanil, flutriafol, folpet,fosetyl-aluminum, fuberidazole, furalaxyl, furametpyr, hexaconazole,hymexazole, guazatine, imazalil, imibenconazole, iminoctadine, iodicarb,ipconazole, iprobenfos, iprodione, iprovalicarb, isoconazole,isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, mandipropamid,maneb, mapanipyrin, mefenoxam, mepronil, metalaxyl, metconazole,methasulfocarb, metiram, metominostrobin/fenominostrobin, mepanipyrim,metrafenone, miconazole, myclobutanil, neo-asozin (ferricmethanearsonate), nuarimol, octhilinone, ofurace, orysastrobin,oxadixyl, oxolinic acid, oxpoconazole, oxycarboxin, paclobutrazol,penconazole, pencycuron, penthiopyrad, pefurazoate, phosphonic acid,phthalide, picobenzamid, picoxystrobin, polyoxin, probenazole,prochloraz, procymidone, propamocarb, propamocarb-hydrochloride,propiconazole, propineb, proquinazid, prothioconazole, pyraclostrobin,pyrazophos, pyrifenox, pyrimethanil, pyrifenox, pyrolnitrine,pyroquilon, quinconazole, quinoxyfen, quintozene, silthiofam,simeconazole, spiroxamine, streptomycin, sulfur, tebuconazole,techrazene, tecloftalam, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate, thiophanate-methyl, thiram, tiadinil,tolclofos-methyl, tolyfluranid, triadimefon, triadimenol, triarimol,triazoxide, tridemorph, trimoprhamide tricyclazole, trifloxystrobin,triforine, triticonazole, uniconazole, validamycin, vinclozolin, zineb,ziram, and zoxamide; nematocides such as aldicarb, oxamyl andfenamiphos; bactericides such as streptomycin; acaricides such asamitraz, chinomethionat, chlorobezilate, cyhexatin, dicofol, dienochlor,etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate,hexythiazox, propargite, pyridaben and tebufenpyrad; and biologicalagents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. The weight ratios of these various mixing partners toother compositions (e.g., herbicides) used in the methods typically arebetween 100:1 and 1:100, or between 30:1 and 1:30, between 10:1 and1:10, or between 4:1 and 1:4.

Further provide are compositions comprising a biologically effectiveamount of a herbicide of interest or a mixture of herbicides, and aneffective amount of at least one additional biologically active compoundor agent and can further comprise at least one of a surfactant, a soliddiluent or a liquid diluent. Examples of such biologically activecompounds or agents are: insecticides such as abamectin, acephate,acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin,azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran,chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl,chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin,lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan,emamectin, endosulfan, esfenvalerate, ethiprole, fenothicarb,fenoxycarb, fenpropathrin, fenvalerate, fipronil, flonicamid,flucythrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron,fonophos, halofenozide, hexaflumuron, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaldehyde, methamidophos,methidathion, methomyl, methoprene, methoxychlor, monocrotophos,methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007), oxamyl,parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet,phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl,pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,trichlorfon and triflumuron; fungicides such as acibenzolar,azoxystrobin, benomyl, blasticidin-S, Bordeaux mixture (tribasic coppersulfate), bromuconazole, carpropamid, captafol, captan, carbendazim,chloroneb, chlorothalonil, copper oxychloride, copper salts,cyflufenamid, cymoxanil, cyproconazole, cyprodinil,(S)-3,5-dichloro-N-(3-chloro-1-ethyl-1methyl-2-oxopropyl)-4-methylbenzamide (RH 7281), diclocymet (S-2900),diclomezine, dicloran, difenoconazole,(S)-3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3-(phenyl-amino)-4H-imidazol-4-one(RP 407213), dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dodine, edifenphos, epoxiconazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid (SZX0722), fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, fluazinam, fludioxonil,flumetover (RPA 403397), flumorf/flumorlin (SYP-L190), fluoxastrobin(HEC 5725), fluquinconazole, flusilazole, flutolanil, flutriafol,folpet, fosetyl-aluminum, furalaxyl, furametpyr (S-82658), hexaconazole,ipconazole, iprobenfos, iprodione, isoprothiolane, kasugamycin,kresoxim-methyl, mancozeb, maneb, mefenoxam, mepronil, metalaxyl,metconazole, metomino-strobin/fenominostrobin (SSF-126), metrafenone(AC375839), myclobutanil, neo-asozin (ferric methane-arsonate),nicobifen (BAS 510), orysastrobin, oxadixyl, penconazole, pencycuron,probenazole, prochloraz, propamocarb, propiconazole, proquinazid(DPX-KQ926), prothioconazole (JAU 6476), pyrifenox, pyraclostrobin,pyrimethanil, pyroquilon, quinoxyfen, spiroxamine, sulfur, tebuconazole,tetraconazole, thiabendazole, thifluzamide, thiophanate-methyl, thiram,tiadinil, triadimefon, triadimenol, tricyclazole, trifloxystrobin,triticonazole, validamycin and vinclozolin; nematocides such asaldicarb, oxamyl and fenamiphos; bactericides such as streptomycin;acaricides such as amitraz, chinomethionat, chlorobezilate, cyhexatin,dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide,fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben andtebufenpyrad; and biological agents including entomopathogenic bacteria,such as Bacillus thuringiensis subsp. Aizawai, Bacillus thuringiensissubsp. Kurstaki, and the encapsulated delta-endotoxins of Bacillusthuringiensis (e.g., Cellcap, MPV, MPVII); entomopathogenic fungi, suchas green muscardine fungus; and entomopathogenic virus includingbaculovirus, nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; andgranulosis virus (GV) such as CpGV. Methods may also comprise the use ofplants genetically transformed to express proteins toxic to invertebratepests (such as Bacillus thuringiensis delta-endotoxins). In suchembodiments, the effect of exogenously applied invertebrate pest controlcompounds may be synergistic with the expressed toxin proteins.

General references for these agricultural protectants include ThePesticide Manual, 13th Edition, C. D. S. Tomlin, Ed., British CropProtection Council, Farnham, Surrey, U.K., 2003 and The BioPesticideManual, 2^(nd) Edition, L. G. Copping, Ed., British Crop ProtectionCouncil, Farnham, Surrey, U.K., 2001.

In certain instances, combinations with other invertebrate pest controlcompounds or agents having a similar spectrum of control but a differentmode of action will be particularly advantageous for resistancemanagement. Thus, compositions can further comprise a biologicallyeffective amount of at least one additional invertebrate pest controlcompound or agent having a similar spectrum of control but a differentmode of action. Contacting a plant genetically modified to express aplant protection compound (e.g., protein) or the locus of the plant witha biologically effective amount of a compound can also provide a broaderspectrum of plant protection and be advantageous for resistancemanagement.

Thus, methods can employ a herbicide or herbicide combination and mayfurther comprise the use of insecticides and/or fungicides, and/or otheragricultural chemicals such as fertilizers. The use of such combinedtreatments can broaden the spectrum of activity against additional weedspecies and suppress the proliferation of any resistant biotypes.

Methods can further comprise the use of plant growth regulators such asaviglycine, N-(phenylmethyl)-1H-purin-6-amine, ethephon, epocholeone,gibberellic acid, gibberellin A₄ and A₇, harpin protein, mepiquatchloride, prohexadione calcium, prohydrojasmon, sodium nitrophenolateand trinexapac-methyl, and plant growth modifying organisms such asBacillus cereus strain BP01.

In further embodiments, methods and compositions for increasing yield ina plant are provided. Specifically, soybean plants having the 3560.4.3.5event and further comprising a second sequence which encodes apolypeptide that imparts tolerance to glyphosate via a distinct mode ofaction from the glyphosate N-acetyltransferase are provided. Such plantsproduce an increase in yield in the presence of an effective amount ofglyphosate when compared to an appropriate control plant. Accordingly,further provided are various methods of increasing yield employing suchplants.

As used herein, the term “yield” refers to the measurable produce ofeconomic value from a crop. This term may be defined in terms ofquantity and/or quality. As used herein, the term “improved yield” meansany improvement in the yield of any measured plant product when comparedto an appropriate control. The improvement in yield can comprise anincrease between about 0.1% to about 90%, about 0.5% to about 10%, about10% to about 20%, about 20% to about 30%, about 30% to about 40%, about40% to about 50%, about 50% to about 60%, about 60% to about 70%, about70% to about 80%, about 80% to about 90% or greater increase in measuredplant product. In other embodiments, the increase in yield can compriseat least a 0.1%. 0.5%, 1%, 3%, 5%. 10%, 15%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or greater increase in the measured plant product.Alternatively, the improved plant yield can comprise between a 0.1 foldto 64 fold, about a 0.1 fold to about a 10 fold, about a 10 fold toabout a 20 fold, about a 20 fold to about a 30 fold, about a 30 fold toabout a 40 fold, about a 40 fold to about a 50 fold, about a 50 fold toabout a 60 fold, about a 60 fold to about a 64 fold increase in measuredplant products.

An improved yield relative to a proper control plant can be measured as(i) increased biomass (weight) of one or more parts of a plant,including aboveground parts or increased biomass of any otherharvestable part; (ii) increased seed yield, which includes an increasein seed biomass (seed weight) and which may be an increase in the seedweight per plant or on an individual seed basis or an increase in seedweight per hectare or acre; (iii) increased number of flowers (florets)per panicle, which is expressed as a ratio of the number of filled seedsover the number of primary panicles; (iv) increased number of (filled)seeds; (v) increased fill rate of seeds (which is the number of filledseeds divided by the total number of seeds and multiplied by 100); (vi)increased seed size, which may also influence the composition of seeds;(vii) increased seed volume, which may also influence the composition ofseeds (for example due to an increase in amount or a change in thecomposition of oil, protein or carbohydrate); (viii) increased seedarea; (ix) increased seed length; (x) increased seed width; (x)increased seed perimeter; (xi) increased harvest index, which isexpressed as a ratio of the yield of harvestable parts, such as seeds,over the total biomass; and (xii) increased thousand kernel weight(TKW), which is extrapolated from the number of filled seeds counted andtheir total weight. An increased TKW may result from an increased seedsize and/or seed weight and may also result from an increase in embryosize and/or endosperm size. For example, an increase in the bu/acreyield of corn derived from a crop having sequence that confer amulti-mode of action glyphosate tolerance as compared with the bu/acreyield from corn having only one of the glyphosate tolerant sequencescultivated under the same conditions would be considered an improvedyield.

A “glyphosate-tolerance polypeptide” is a polypeptide that confersglyphosate tolerance on a plant (i.e., that makes a plantglyphosate-tolerant), and a “glyphosate-tolerance polynucleotide” is apolynucleotide that encodes a glyphosate-tolerance polypeptide. “Mode ofaction” refers to the specific metabolic or physiological process withinthe plant by which the glyphosate-tolerant polypeptide acts to protectthe plant from glyphosate. Thus, polypeptides having “distinct” modes ofaction for providing glyphosate tolerance comprise any two or morepolypeptides that protect a plant from glyphosate by a number ofmechanisms including detoxifying the chemical via different metabolic orphysiological processes. For example, glyphosate N-acetyl transferasepolypeptides acetylate glyphosate and thereby detoxify the herbicide,while glyphosate-tolerant EPSPS polypeptides prevent or decrease theability of glyphosate to inhibit the shikimic acid pathway. In light ofthe distinct mechanism of action of these two enzymes, thesepolypeptides represent two non-limiting examples of polypeptides thatconfer tolerance to glyphosate via distinct modes of action.

Methods of increasing yield comprise planting the area of cultivationwith the multi-mode of action glyphosate-tolerant 3560.4.3.5 soybeanseeds or plants, and applying to any of the soybean plant, soybean part,weed or area of cultivation thereof an effective amount of glyphosate.It is recognized that the herbicide can be applied before or after thecrop is planted in the area of cultivation. A “control” or “controlplant” or “control plant cell” provides a reference point for measuringchanges in phenotype (i.e., improved yield) of the subject plant orplant cell, and may be any suitable plant or plant cell. An improvedyield can be evaluated by statistical analysis of suitable parameters.The plant being evaluated is referred to as the “test plant.” Typically,when measuring an improved yield, an appropriate control plant is onethat expresses one of the glyphosate-tolerance sequences that is presentin the test plant but lacks or does not express additional (second,third, etc.) glyphosate-tolerance sequences in the test plant. Forexample, in evaluating multi-mode of action glyphosate-tolerant3560.4.3.5 soybean plant for an increased yield, an appropriate controlplant would be a plant that expresses GLYAT and not EPSPS or one thatexpresses EPSPS and not GLYAT, or one that expresses GLYAT and notglyphosate oxido-reductase or one that expresses glyphosateoxido-reductase and not GLYAT. One skilled in the art will be able todesign, perform, and evaluate a suitable controlled experiment to assessthe glyphosate tolerance of a plant of interest and the improved yield,including the selection of appropriate test plants, control plants, andtreatments.

The improved yield of the multi-mode of action glyphosate-tolerant3560.4.3.5 soybean plant can be assessed at various times after a planthas been treated with the glyphosate. Improved yield is ultimatelydetermined as productivity relative for the product (fresh cut weight,silage yield, mature grain harvest). Improved yield determination canoccur at any stage of maturity of the test plant by assessing yieldcomponent measures. Any time of assessment is suitable as long as itpermits detection of an improved yield of test plants as compared to thecontrol plants. Flower number could be measured at R2. Plant biomasscould be measured at anytime during the growing season but measurementswould be applicable to only that exact point in crop stage. Seed yield,seed size, and seed number is reliably measured at crop growth stage R7and R8. In the case of crops such as vegetables, plant fresh weight isdetermined at or before peak produce harvest.

As used herein, an “effective amount of glyphosate” in reference toimproving yield is one that is sufficient to improve the yield in theplants having the glyphosate-tolerant sequences which act via twodistinct modes of action and further comprises an amount that istolerated by the plant, and in specific embodiments, the effectiveamount is further capable of controlling weeds in the area ofcultivation. It is further recognized that when the multi-mode of actionglyphosate tolerant plants further comprises additional traits thatimpart tolerance to other herbicides, the methods of the invention cancomprise applying to such plants glyphosate plus an additionalappropriate herbicide. In such cases, an “effective amount of aherbicide” is one that is tolerated by the plant and controls weeds inthe area of cultivation.

“Herbicide-tolerant” or “tolerant” or “crop tolerance” in the context ofherbicide or other chemical treatment as used herein means that a plantor other organism treated with a particular herbicide or class orsubclass of herbicide or other chemical or class or subclass of otherchemical will show no significant damage or less damage following thattreatment in comparison to an appropriate control plant.

Thus, a plant is tolerant to a herbicide if it shows damage incomparison to an appropriate control plant that is less than the damageexhibited by the control plant by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%,250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more. Inthis manner, a plant that is tolerant to a herbicide or other chemicalshows “improved tolerance” in comparison to an appropriate controlplant. Damage resulting from herbicide or other chemical treatment isassessed by evaluating any parameter of plant growth or well-beingdeemed suitable by one of skill in the art. Damage can be assessed byvisual inspection and/or by statistical analysis of suitable parametersof individual plants or of a group of plants. Thus, damage may beassessed by evaluating, for example, parameters such as plant height,plant weight, leaf color, leaf length, flowering, fertility, silking,yield, seed production, and the like. Damage may also be assessed byevaluating the time elapsed to a particular stage of development (e.g.,silking, flowering, or pollen shed) or the time elapsed until a planthas recovered from treatment with a particular chemical and/orherbicide.

In making such assessments, particular values may be assigned toparticular degrees of damage so that statistical analysis orquantitative comparisons may be made. The use of ranges of values todescribe particular degrees of damage is known in the art, and anysuitable range or scale may be used. For example, herbicide injuryscores (also called tolerance scores) can be assigned using the scaleset forth are known in the art.

By “no significant damage” is intended that the concentration ofherbicide either has no effect on the plant or when it has some effecton a plant from which the plant later recovers, or when it has an effectwhich is detrimental but which is offset, for example, by the impact ofthe particular herbicide on weeds. Thus, for example, a crop plant isnot “significantly damaged by” a herbicide or other treatment if itexhibits less than 50%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or 1% decrease in at least one suitable parameterthat is indicative of plant health and/or productivity in comparison toan appropriate control plant (e.g., an untreated crop plant). Suitableparameters that are indicative of plant health and/or productivityinclude, for example, plant height, plant weight, leaf length, timeelapsed to a particular stage of development, flowering, yield, seedproduction, and the like. The evaluation of a parameter can be by visualinspection and/or by statistical analysis of any suitable parameter.Comparison may be made by visual inspection and/or by statisticalanalysis. Accordingly, a crop plant is not “significantly damaged by” aherbicide or other treatment if it exhibits a decrease in at least oneparameter but that decrease is temporary in nature and the plantrecovers fully within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6weeks.

Conversely, a plant is significantly damaged by a herbicide or othertreatment if it exhibits more than a 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%,145%, 150%, or higher decrease in at least one suitable parameter thatis indicative of plant health and/or productivity in comparison to anappropriate control plant (e.g., an untreated weed of the same species).Thus, a plant is significantly damaged if it exhibits a decrease in atleast one parameter and the plant does not recover fully within 1 week,2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks.

Glyphosate can be applied to the multi-mode of actionglyphosate-tolerant 3560.4.3.5 soybean plants or their area ofcultivation. Non-limiting examples of glyphosate formations are setforth above in Table 2. In specific embodiments, the glyphosate is inthe form of a salt, such as, ammonium, isopropylammonium, potassium,sodium (including sesquisodium) or trimesium (alternatively namedsulfosate).

In some embodiments, a method of improving yield in a multi-mode ofaction glyphosate-tolerant 3560.4.3.5 soybean plant comprises atreatment with the glyphosate applied to that plant at a dose equivalentto a rate of at least 210, 420, 840, 1260, 1680, 2100, 2520, 2940, 3360,3780, 4200, 4620, 5040, 5460, 5880, 6300, 6720, or more grams of acidequivalent of glyphosate in a commercial herbicide formulation herbicideper hectare.

In other embodiments, glyphosate is applied to an area of cultivationand/or to at least one multi-mode of action glyphosate tolerant3560.4.3.5 soybean plant in an area of cultivation at rates between 210and 3360, between 210 and 500, between 500 and 1000, between 1000 and1500, between 1500 and 2000, between 2000 and 2500, between 2500 and3000, and between 3000 and 3360 grams acid equivalent per hectare at thelower end of the range of application and between 3780 and 6720, 3780and 4000, between 4000 and 4500, between 4500 and 5000, between 5000 and5500, between 5500 and 6000, between 6500 and 6720 grams of acidequivalent per hectare at the higher end of the range of application. Inone embodiment, the range of glyphosate application for soybean is asingle dose of up to 1680 grams acid equivalent per hectare, and a fullin-crop season dose up to 2520 grams acid equivalent per hectare.

Methods to improve yield allow for the application of glyphosatepre-plant, at plant or any time after planting multi-mode of actionglyphosate tolerant 3560.4.3.5 soybean seeds are planted in an area ofcultivation. Such timing of applications are discussed in further detailelsewhere herein. The time at which glyphosate is applied may bedetermined with reference to the size of plants and/or the stage ofgrowth and/or development of plants in the area of interest, e.g., cropplants or weeds growing in the area. The stages of growth and/ordevelopment of soybean plants are known in the art and are discussed infurther detail elsewhere herein. Thus, for example, the time at whichglyphosate is applied to an area of interest which plants are growing toincrease the yield of the plant may be the time at which some or all ofthe plants in a particular area have reached at least a particular sizeand/or stage of growth and/or development, or the time at which some orall of the plants in a particular area have not yet reached a particularsize and/or stage of growth and/or development.

As discussed above, the multi-mode of action glyphosate-tolerant3560.4.3.5 soybean plant can further comprise sequences that imparttolerance to additional herbicides. Thus, depending on the additionalsequences present in the plant, the methods of the invention can furthercomprise applying additional herbicides of interest to the plant andthereby improve yield and control weeds in an area of cultivation. Thus,as discussed above, the methods of the invention encompass the use ofsimultaneous and/or sequential applications of multiple classes ofherbicides.

When glyphosate is used with additional herbicides of interest, theapplication of the herbicide combination need not occur at the sametime. So long as the field in which the crop is planted containsdetectable amounts of the first herbicide and the second herbicide isapplied at some time during the period in which the crop is in the areaof cultivation, the crop is considered to have been treated with amixture of herbicides according to the invention.

The classifications of herbicides (i.e., the grouping of herbicides intoclasses and subclasses) is well-known in the art and includesclassifications by HRAC (Herbicide Resistance Action Committee) and WSSA(the Weed Science Society of America) (see also, Retzinger andMallory-Smith (1997) Weed Technology 11: 384-393). An abbreviatedversion of the HRAC classification (with notes regarding thecorresponding WSSA group) is set forth below in Table 2. A morecomprehensive list of specific herbicides can be found for example, inU.S. Application Publication 2007/0130641, herein incorporated byreference. In some embodiments, when additional herbicides are appliedto increase yield, the rate of application will be sufficient to controlweeds and such rates of application are disclosed elsewhere herein.

In non-limiting embodiments, the multi-mode of actionglyphosate-tolerant 3560.4.3.5 soybean plant comprises a sequenceencoding a glyphosate N-acetyl transferase polypeptide and an EPSPSpolypeptide, where the plant or the area of cultivation is treated withan effective amount of glyphosate to thereby improve the yield of theplant. Such methods to improve yield can comprise applying to the plantor area of cultivation an effective amount of glyphosate to therebyimprove the yield of said plant and further applying an effectiveconcentration of an additional herbicide, such as an ALS chemistry, toeffectively control the weeds in said area of cultivation. Since ALSinhibitor chemistries have different herbicidal attributes, blends ofALS inhibitors plus other chemistries can provide superior weedmanagement strategies including varying and increased weed spectrum, theability to provide specified residual activity (SU/ALS inhibitorchemistry with residual activity leads to improved herbicidal activitywhich leads to a wider window between glyphosate applications, as wellas, an added period of control if weather conditions prohibit timelyapplication).

Embodiments are further defined in the following Examples. It should beunderstood that these Examples are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics, and without departing from thespirit and scope thereof, can make various changes and modifications ofthe embodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

EXPERIMENTAL Example 1 Insert and Flanking Border SequenceCharacterization of Soybean Event 3560.4.3.5

Soybean event 3560.4.3.5, hereafter referred to as 3560.4.3.5 soybean,was obtained by microprojectile bombardment with the Not I-Asc Ifragment from plasmid PHP20163. This fragment, PHP20163A, contains theglyphosate acetyltransferase (glyat 4601) gene under the control of theSCP1 promoter that is a synthetic constitutive promoter comprising aportion of the CaMV 35S promoter (Odell et al. (1985) Nature313:810-812) and the Rsyn7-Syn II Core synthetic consensus promoter(U.S. Pat. Nos. 6,072,050 and 6555673). See also, for example,US20030226166, Table 13, and SEQ ID NO:3. Downstream of this element isthe Tobacco Mosaic Virus (TMV) omega 5′UTR translational enhancerelement (Gallie et al. (1992) Nucleic Acid Research 20:4631-4638) andthe proteinase inhibitor II (pinII) terminator from Solanum tuberosum(Keil et al. (1986) Nucleic Acid Research 14:5641-5650 and An et al.(1989) Plant Cell 1:115-122). PHP20163A also contains the gm-hra genethat is a modified version of the acetolactate synthase gene fromsoybean with 15 additional nucleotides on the 5′ end (2697-2711) derivedfrom the als 5′ UTR and two nucleotides changes within the codingsequence under the control of the S-adenosyl-L-methionine synthetase(SAMS) promoter (US Application Publication 2003/0226166) with its 5′UTRand intron (SAMS Pro) and the acetolactate synthase (gm-als) terminator,both from soybean.

To characterize the integrity of the inserted DNA from PHP20163A and thegenomic insertion site, the sequence of the insert and flanking genomicDNA border regions of 3560.4.3.5 soybean was determined. In total, 10849base pairs (bp) of 3560.4.3.5 soybean was sequenced, comprising 5362 bpof DNA insert from PHP20163A, 3317 bp of 5′ flanking genomic bordersequence, and 2170 bp of 3′ flanking genomic border sequence. Whencompared to the expected sequence from the DNA fragment used fortransformation, the insert was confirmed to be intact and identical toPHP20163A and precisely integrated into the soy genome. PCRamplification from the 3560.4.3.5 soybean insert and border sequencesconfirmed that the border regions were of soybean origin and that thejunction regions could be used for identification of 3560.4.3.5 soybean.

BLASTn analysis of the border regions resulted in significant identitiesto public and proprietary soybean genomic sequences. Overall,characterization of the insert and genomic border sequence of 3560.4.3.5soybean along with Southern blot data (data not show) indicated that asingle insertion of the DNA fragment, PHP20613A, was present in thesoybean genome.

The following abbreviations are used in describing the presentinvention.

ALS acetolactate synthase protein bp base pair glyat glyphosateacetyltransferase gene GLYAT glyphosate acetyltransferase proteinGLYAT4601 glyphosate acetyltransferase protein derived from glyat4601gene gm-als wild type acetolactate synthase gene from soybean gm-hramodified version of acetolactate synthase gene from soybean kb kilobasePCR polymerase chain reaction UTR untranslated region als acetolactatesynthase gene AMS ammonium sulfate DAT days after treatment glyat4601glyphosate acetyl transferase gene from the 7^(th) round of DNAshuffling a glyat gene family isolated from Bacillus licheniformis(Castle et al. (2004) Science 304: 1151-1154; Siehl et al. (2005) PestManag. Sci. 61: 235-240). GLYAT4601 glyphosate acetyl transferaseprotein from the 7^(th) round of DNA shuffling a glyat gene familyisolated from Bacillus licheniformis (Castle et al. (2004) Science 304:1151-1154; Siehl et al. (2005) Pest Manag. Sci. 61: 235-240). hra highlyresistant allele of the acetolactate synthase gene glyat + gm-hratransgenic event expressing both the glyat4601 and gm-hra genes NILsnear isogenic lines NIS non-ionic surfactant SAMSS-adenosyl-L-methionine synthase promoter (U.S. patent application No.2003/0226166) SCP1 Synthetic constitutive promoter 1 (U.S. Pat. Nos.6,072,050 and 6,555,673 B1.

Soybean (Glycine max) has been modified by the insertion of theglyphosate acetyltransferase (glyat4601) gene derived from Bacilluslicheniformis and a modified version of the soybean acetolactatesynthase gene (gm-hra). The glyat4601 gene was functionally improved bya gene shuffling process to optimize the kinetics of glyphosateacetyltransferase (GLYAT) activity for acetylating the herbicideglyphosate. The insertion of the glya4601t gene in the plant conferstolerance to the herbicidal active ingredient glyphosate through theconversion of glyphosate to the non-toxic acetylated form. The insertionof the gm-hra gene produces a modified form of the acetolactate synthaseenzyme. ALS is essential for branched chain amino acid biosynthesis andthe modification in the gm-hra gene overcomes this inhibition and thusprovides tolerance to a wide range of ALS-inhibiting herbicides.

The publicly available cultivar Jack was used as the recipient line forgeneration of 3560.4.3.5 soybean. The 3560.4.3.5 soybean was obtained bymicroprojectile bombardment with the Not I-Asc I fragment from plasmidPHP20163 (FIG. 1). This fragment, PHP20163A (FIG. 2), contains theglyat4601 gene under the control of the SCP1 promoter and Tobacco MosaicVirus (TMV) omega 5′ UTR translational enhancer element and theproteinase inhibitor II (pinII) terminator from Solanum tuberosum.PHP20163A also contains the gm-hra gene under the control of theS-adenosyl-L-methionine synthetase (SAMS) promoter and the acetolactatesynthase (gm-als) terminator, both from soybean.

The transgenic 3560.4.3.5 soybean was generated using the BiolisticsPDS-1000/He particle gun, manufactured by Bio-Rad (Hercules, Calif.),essentially as described by Klein et al. (1987). The targets fortransformation were clumps of secondary somatic embryos derived fromexplants from small, immature soybean seeds of the cultivar Jack. Thesecondary somatic embryos were excised from immature explants,transferred to a liquid soybean culture maintenance medium, andsubcultured at regular intervals until prepared for bombardment.

Soybean somatic embryogenic cultures were used in transformationexperiments 2-4 months after initiation. On the day of transformation,microscopic gold particles were coated with the purified fragmentPHP20163A DNA and accelerated into the embryogenic soybean cultures.Only PHP20163A DNA was used, and no additional DNA (e.g., carrier DNA)was used in the transformation process.

Following the transformation, the soybean tissue was transferred toflasks of fresh liquid culture maintenance medium for recovery. Afterseven days, the liquid culture medium was changed to culture maintenancemedium supplemented with chlorsulfuron as the selection agent.Chlorsulfuron belongs to a family of ALS-inhibiting herbicides, andtherefore only soybean cells that had stably inherited the gm-hratransgene continued to grow.

After several weeks in the culture maintenance medium supplemented withchlorsulfuron, small islands of healthy, chlorsulfuron-tolerant greentissue became visible and started to grow out of pieces of dying somaticembryogenic tissue. Green embryogenic clumps were excised fromassociated pieces of dying or dead tissue and received regular changesof fresh liquid selection medium until the start of the regenerationprocess. Embryogenic tissue samples were analyzed to confirm thepresence of the glyat4601 and gm-hra transgenes by Southern blothybridization. TO plants were regenerated and transferred to thegreenhouse for seed production. FIG. 5 describes a breeding diagram.

In the microprojectile bombardment transformation the 3560.4.3.5soybean, DNA is inserted into the plant genome. The integration of theDNA fragment can occur at virtually any site in the plant genome. Onceinserted, the genes that contain plant expression elements arerecognized by the plant and may be expressed. Various moleculartechniques are then used to specifically characterize the integrationsite in the 3560.4.3.5 soybean.

Southern blot analyses indicated that a single, intact PHP20163Afragment inserted into the soybean genome to produce the 3560.4.3.5soybean (data not shown). Cloning and sequencing of the flanking genomicborder regions of 3560.4.3.5 soybean and the inserted DNA was undertakento characterize the insertion site in the soybean genome and obtainsequence that could be used to uniquely identify 3560.4.3.5 soybean.

Leaf tissue from the T5 generation of 3560.4.3.5 soybean was used as foradditional sequence characterization. The T5 generation representstransformation of a Jack soybean variety, followed by twoself-crossings. A single plant from this second self-crossing wasselected from three subsequent rounds of self-crossing and seed bulking.Southern blot analysis was used for event confirmation on plant leaftissue of 3560.4.3.5 soybean (data not shown).

Leaf tissue from soybean plants that were not genetically modified wasused as a control for sequence characterization. The unmodified soybeanplants have a genetic background representative of 3560.4.3.5 soybeanbackground; however, they do not contain the plant transcription unitsfor the glyat4601 and gm-hra genes.

The 100 bp and 1 kb step DNA Ladders (Promega, Madison, Wis.) were usedto estimate DNA fragment sizes on agarose gels.

Soybean seed for 3560.4.3.5 soybean and unmodified control seed wereplanted to produce sufficient numbers of plants for DNA analysis. Forcharacterization of 3560.4.3.5 soybean line, eight T5 seeds wereplanted. Eight seeds were planted for control soybean line as well. Oneseed was planted per pot, and the pot was uniquely identified. Plantingand growing conditions were conducive to healthy plant growth includingregulated light and water.

Leaf samples were collected for each of the control and 3560.4.3.5soybean plants. For each sample, sufficient leaf material from above thegrowing point was collected and placed in a pre-labeled sample bag. Thesamples were placed on dry ice and were transferred to an ultra lowfreezer following collection. All samples were maintained frozen untilprocessing.

Frozen leaf samples (1-2 gram quantities) were ground, and the genomicDNA was isolated using a modified Urea Extraction Buffer procedure (Chenet al. (1994) Urea-based plant DNA miniprep in Freeling M. and WalbotV., eds, The Maize Handbook, Springer-Verlag, New York, p 526-527).Genomic DNA was extracted from leaf tissue harvested from individualplants as described above. Specifically, the tissue was pulverized intubes containing grinding beads using a Geno/Grinder™ (SPEX CertiPrep,Inc., Metuchen, N.J.) instrument and the genomic DNA isolated using astandard procedure. Approximately 1 gram ground tissue was extractedwith 5 mL Urea Extraction Buffer (7 M Urea, 0.34 M NaCl, 0.05 MTris-HCl, pH 8.0, 0.02 M EDTA, 1% N-Lauroylsarcosine) for 12-30 minutesat 37° C., followed by two extractions with phenol/chloroform/isoamylalcohol (25:24:1) and one extraction with water saturated chloroform.The DNA was precipitated from the aqueous phase by the addition of 1/10volume of 3 M NaOAc (pH 5.2) and 1 volume of isopropyl alcohol, followedby centrifugation to pellet the DNA. After washing the pellet twice with70% ethanol, the DNA was dissolved in 0.5 mL TE buffer (10 mM Tris, 1 mMEDTA, pH 7.5) and treated with 10 μg Ribonuclease A for 15 minutes at37° C. The sample was extracted once with phenol:chloroform:isoamylalcohol (25:24:1) and once with water saturated chloroform, followed byprecipitation with isopropyl alcohol and washing with 70% ethanol. Afterdrying, the DNA was re-dissolved with 0.5 mL TE buffer and stored at 4°C. Following extraction, the DNA was visualized on an agarose gel todetermine the DNA quality, and was quantified using Pico Green® reagent(Molecular Probes, Inc., Eugene, Oreg.) and spectrofluorometricanalysis.

Phenotypic analysis of 3560.4.3.5 soybean plants and control plants wascarried out by western blot analysis using antibodies to the GLYAT4601protein to confirm the absence or presence of the GLYAT4601 protein inmaterial used for Southern blot analysis and sequence characterization.Total protein was extracted by grinding several leaf punches tohomogeneity in 150 μl of protein extraction buffer (50 mM Tris-HCl (pH7.5), 0.1% SDS, and 10 mM β-mercaptoethanol). An aliquot of each crudeextract was mixed with LDS Sample Buffer and reducing agent (Invitrogen)and heated to approximately 95° C. for 5 minutes. Proteins wereseparated by size under denaturing conditions through a NuPAGE™ Bis/TrisGel system as described (Invitrogen). Selected molecular weightstandards were used to determine sufficient migration in the gel and formolecular weight determination on the western blot (Invitrogen). TheBis/Tris gel was transferred to a nitrocellulose membrane using themethod as described for the Novex™ XCell II™ Blot Module WesternTransfer (Invitrogen). Alternatively, gels were stained with SimplyBlue™SafeStain (Invitrogen) to visualize the proteins to verify equivalentsample loading.

The GLYAT4601 protein band was detected using the WesternBreeze®Chemiluminescent Western Blot Immunodetection Kit as described(Invitrogen). Primary monoclonal antibodies specific for the GLYAT4601protein were used with the WesternBreeze® Kit. Bands were thenvisualized using a chemiluminescent substrate. Blots were exposed toX-ray film for one or more time points to detect protein bands. PurifiedGLYAT4601 protein was used as a positive control on the western blots.Plants were scored as positive for GLYAT4601 when a band of theappropriate size was present and scored as negative when the band wasabsent on the western blots.

A preliminary Southern blot analysis of DNA isolated from all 3560.4.3.5soybean plants was used to verify the presence of both the glyat4601 andgm-hra genes. Methods for this preliminary characterization aredescribed below. Final Southern blot analysis was carried out on asubset of 3560.4.3.5 soybean plants (data not shown).

Genomic DNA samples extracted from selected 3560.4.3.5 soybean andcontrol soybean plants were digested with restriction enzymes followinga standard procedure. Approximately 2 μg of genomic DNA was digested ina volume of 100 μL using 50 units of enzyme according to manufacturer'srecommendations. The digestions were carried out at 37° C. for threehours, followed by ethanol precipitation with 1/10 volume of 3 M NaOAc(pH 5.2) and 2 volumes of 100% ethanol. After incubation at 4° C. andcentrifugation, the DNA was allowed to dry and re-dissolved in TEbuffer. The reference plasmid, PHP20163, was spiked into a control plantDNA sample in an amount equivalent to approximately one or three genecopies per soybean genome and digested with the same enzyme to serve asa positive control for probe hybridization and to verify sizes ofinternal fragments on the Southern blot.

Following restriction enzyme digestion, the DNA fragments produced wereelectrophoretically separated by size through an agarose gel and amolecular weight standard ΦX174 RF DNA/Hae III Fragments (Invitrogen)was used to determine sufficient migration and separation of thefragments on the gel. DIG labeled DNA Molecular Weight Marker VII(Roche), visible after DIG detection as described below, was used todetermine hybridizing fragment size on the Southern blots.

Agarose gels containing the separated DNA fragments were depurinated,denatured, and neutralized in situ, and transferred to a nylon membranein 20×SSC buffer (3M NaCl, 0.3 M Sodium Citrate) using the method asdescribed for the TURBOBLOTTER™ Rapid Downward Transfer System(Schleicher & Schuell, Keene, N.H.). Following transfer to the membrane,the DNA was bound to the membrane by UV crosslinking (Stratalinker,Stratagene, La Jolla, Calif.). Probes for the SCP1 promoter, glyat4601,pinII terminator, SAMS, gm-hra, and als terminator were used to detectgenes and elements within the insertion (Table 3). Backbone andhygromycin resistance gene cassette regions (backbone 20163 and hyg20163probes) of the PHP20163 plasmid were used to verify absence of plasmidbackbone DNA in 3560.4.3.5 soybean (Table 3). DNA fragments of the probeelements were generated by PCR from plasmid PHP20163 (FIG. 1) or aplasmid with equivalent elements using specific primers. PCR fragmentswere electrophoretically separated on an agarose gel, excised andpurified using a gel purification kit (Qiagen, Valencia, Calif.). DNAprobes were generated from these fragments by PCR that incorporated aDIG labeled nucleotide, [DIG-11]-dUTP, into the fragment. PCR labelingof isolated fragments was carried out according to the proceduressupplied in the PCR DIG Probe Synthesis Kit (Roche).

TABLE 3 Description of DNA Probes Used for Southern Blot HybridizationPosition on Position on FIG. PHP20163A PHP20163 Length Probe NameGenetic Element Probe (bp to bp) (bp to bp) (bp) SCP1 promoter SCP1promoter FIG. 6 12 to 479 (SEQ  12 to 479 486 probe 1 ID NO: 29)glyat4601 glyat4601 gene FIG. 6 597 to 1012  597 to 1012 416 probe 2(SEQ ID NO: 30) pinII terminator pinII terminator FIG. 6 1107 to 13401107 to 1340 234 probe 3 (SEQ ID NO: 31) SAMS¹ SAMS promoter and FIG. 61702 to 2146 1702 to 2146 445 intron elements probe 4 (SEQ ID NO: 32)2147 to 2638 492 2147 to 2638 (SEQ ID NO: 33) gm-hra¹ gm-hra gene FIG. 62700 to 3629 2700 to 3629 930 probe 5 (SEQ ID NO: 34) 3635 to 4664 10303635 to 4664 (SEQ ID NO: 35) gm-als gm-als terminator FIG. 6 4670 to5318 4670 to 5318 649 terminator probe 6 (SEQ ID NO: 36) backboneplasmid backbone of FIG. 7 N/A² 7427-7954 528 20163¹ PHP20163 probe A6665-7416 752 hyg 20163¹ hygromycin resistance FIG. 7 N/A 6097-6619 523gene of PHP20163 probe B 5389-6091 703 ¹Two non-overlapping segmentswere generated for this probe and were combined for hybridization. ²NotApplicable; these are not present on the PHP20163A fragment.

Genomic DNA isolated from 3560.4.3.5 soybean plants was digested withHind III and Xba I, and electrophoretically separated, transferred tonylon membranes, and hybridized to the glyat4601 and gm-hra gene probes.Labeled probes were hybridized to the target DNA on nylon membranes fordetection of the specific fragments using the procedures essentially asdescribed for DIG Easy Hyb solution (Roche). After stringent washes, thehybridized DIG-labeled probes and DIG-labeled DNA standards werevisualized using CDP-Star Chemiluminescent Nucleic Acid Detection Systemwith DIG Wash and Block Buffer Set (Roche). Blots were exposed to X-rayfilm for one or more time points to detect hybridizing fragments and tovisualize molecular weight standards. Images were then digitallycaptured by detection with the Luminescent Image Analyzer LAS-3000(Fujifilm Medical Systems, Stamford, Conn.). The sizes of detected bandswere documented for each digest and each probe.

Following hybridization and detection, membranes were stripped ofDIG-labeled probe to prepare the blot for subsequent re-hybridization toadditional probes. Membranes were rinsed briefly in distilled,de-ionized water and then stripped in a solution of 0.2 M NaOH and 1.0%SDS at 40° C. with constant shaking. The membranes were then rinsed in2×SSC and either used directly for subsequent hybridizations or storedat 4° C. or −20° C. for later use. The alkali-based stripping procedureeffectively removes probes labeled with the alkali-labile DIG. Thispreliminary Southern blot analysis showed the presence of the insertionin 3560.4.3.5 soybean plants and confirmed that 3560.4.3.5 soybeanplants used for this study contained the same insertion (Table 4).

TABLE 4 Summary of Preliminary Southern Screen Data for 3560.4.3.5Soybean Line. Southern Blot Southern Blot Plant ID Sample ID glyat4601Probe² gm-hra Probe² T-F-05-140S-17 T-17 + + T-F-05-140S-18 T-18 + +T-F-05-140S-19 T-19 + + T-F-05-140S-20 T-20 + + T-F-05-140S-21 T-21 + +T-F-05-140S-22 T-22 + + T-F-05-140S-23 T-23 + + T-F-05-140S-24T-24 + + + indicates hybridization signal on Southern blot.

Four plants were chosen for insert and border sequence analysis:3560.4.3.5 soybean plants T17 and T18, and control plants C1 and C2. PCRprimers were synthesized by Sigma-Genosys, Inc. (The Woodlands, Tex.)and MWG (Ebersberg, Germany), and used at concentrations of 0.3-0.4 uM.For amplification of the PHP20163A insert region, both theAdvantage-GC-2 PCR system (Clontech) and the Expand Long Template PCRSystem (Roche) were used to amplify genomic DNA (100-500 ng); and forthe 5′ and 3′ flanking genomic border PCR, the Advantage-GC-2 PCR systemwas used to amplify from genomic DNA (10 ng). The PCR products werevisualized under UV light following electrophoresis through a 1% agarosegel with 1×TAE and ethidium bromide; excised, and purified from the gelusing Qiaquick gel purification kit (Qiagen, Valencia, Calif.).

The PCR products were cloned using the TOPO TA-cloning Kit (pCR2.1-TOPOvector, Invitrogen, Carlsbad, Calif.). Plasmids were isolated usingQIAprep Spin Miniprep Kit (Qiagen), screened by restriction enzymedigestions, and sequenced.

For complete sequence coverage of the cloned PCR products representingthe insert of the 3560.4.3.5 soybean, the clones were sequenced by atransposon-based sub-cloning method to facilitate bi-directionalsequencing of a cloned insert from the site of the transposition event(MJ Research TGS system; Happa et al. (1999) Nucleic Acid Research27:2777-2784. Since unintended mutations can occur during the generationof the PCR products, we cloned and sequenced products from two separatePCR reactions with T17 and T18 DNA. In all cases, any PCR-inducedsequence error was present in only one of the four clones, allowingreliable consensus calls to be made on every base of the sequence.

Initial sequence characterization of the 5′ flanking border was carriedout using the BD GenomeWalker Universal Kit (Clontech, Mountain View,Calif.). The GenomeWalker protocol involves digesting the genomic DNAwith various restriction enzymes and ligating these digests with anadaptor, thereby creating “libraries” to be used as template for tworounds of PCR. The PCR primers for the 2 rounds of PCR consisted of aninsert-specific (SCP1 promoter) primer, and an adaptor-specific primer.Based on sequence information generated from the Genome Walkerexperiments, primers were designed to perform PCR on genomic DNA from3560.4.3.5 and control soybean plants, using primers that spanned the 5′junction. (FIG. 3 and data not shown). To extend the 5′ border sequencefurther, we used the DNA Walking SpeedUp Kit (Seegene, Inc. Rockville,Md.) was used, which is another PCR-based genomic DNA walking approachbased on the DNA Walking Annealing Control Primer PCR Technology (Ochmanet al. (1988) Genetics 120:621; Silver et al. (1989) Journal of Virology63:1924; Triglia et al. (1988) NAR 16:8186). The last round of borderextension was performed by anchoring within the 5′-most end of theborder and again using the Genome Walker kit. The final 5′ flankingborder sequence was verified by cloning and sequencing of 5′ flankinggenomic border PCR products. In order to demonstrate that the identified5′ flanking genomic border sequence is of soybean origin, PCR wasperformed within this 5′ flanking genomic region on genomic DNA from3560.4.3.5 soybean and control soybean plants (FIG. 3 and data notshown).

Initial sequence characterization of the 3′ flanking border was carriedout using inverse PCR (Silver et al. (1989) Journal of Virology 63:1924;Ochman et al. (1988) Genetics 120:621; Triglia et al. (1988) NAR16:8186), with insert-specific primers anchored in the glyat4601 andgm-hra genes. Sequence obtained from products generated using inversePCR was then used to design primers for PCR on genomic DNA from3560.4.3.5 and control soybean plants, using primers that spanned the 3′junction. (FIG. 3 and data not shown). The 3′ flanking genomic bordersequence was verified by cloning and sequencing the 3′ flanking genomicborder PCR products. The genomic flanking border was extended furtherusing the sequence information from proprietary soybean sequence thatmatched this flanking border to design PCR primers. Cloning andsequencing the resulting PCR products confirmed this additional flankingborder sequence. In order to demonstrate that the identified 3′ flankinggenomic border sequences were of soybean origin, PCR was performedwithin this 3′ genomic region on DNA from 3560.4.3.5 soybean and controlsoybean plants (FIG. 3 and data not shown).

For verification of the DNA sequence that inserted into the soybeangenome, PCR was performed to amplify, clone, and sequence the insertedDNA from 3560.4.3.5 soybean. PCR primers just outside the insert (primerpair 1297/1298) were used to amplify the entire insert from DNA of3560.4.3.5 soybean plants. This amplification produced products of theexpected size (5.4 kb) from two separate reactions from each of two testsamples; these were cloned and sequenced. In addition, the DNA fragmentPHP20163A used for creating 3560.4.3.5 soybean line was sequenced, andcompared with the insert sequence generated from 3560.4.3.5 soybeangenomic DNA.

Both the 5′ and 3′ flanking border sequences of the 3560.4.3.5 soybeanwere subjected to BLASTn analysis in order to identify the nature andpotential function of the flanking sequences in the soybean genome. Thesearches were performed against the NCBI Genbank Nucleotide (“nt”)dataset (www.ncbi.nlm.nih.gov/), Release 154, last updated Jul. 31,2006, 4,302,011 total sequences), as well as to Genome Survey Sequencedataset (GSS, Release Jul. 28, 2006). Finally, sequences were comparedto a dataset consisting of all proprietary soybean genomic and ESTsequences generated by Pioneer. Default parameters were used in allcases. Both 5′ and 3′ border/insert junctions were also screened for thepresence of novel open reading frames (ORFs)≧100 amino acids (300 bp) inlength using Vector NTI 9.1 (Invitrogen, Carlsbad, Calif.).

DNA isolated from eight 3560.4.3.5 soybean plants was digested with XbaI and Hind III, and hybridized to gm-hra and glyat4601 gene probes toverify the presence of the insertion and to verify the molecularequivalence of the insertion among all 8 plants analyzed. Thepreliminary Southern blot screen indicated that 3560.4.3.5 soybeanplants exhibited identical hybridization patterns (Table 4 and data notshown). In the Xba I digests, the glyat4601 probe hybridized to a 1.4 kbband and the gm-hra probe hybridized to a 3.9 kb band as expected (FIG.2), demonstrating the intact insertion of PHP20163A. In the Hind IIIdigests, the glyat4601 probe hybridized to a 5′ flanking 6.1 kb band,the gm-hra probe hybridized to 3′ flanking 7.4 kb band, and an internal2.4 kb band, demonstrating that the insertion exists as a single copy inthe genome (FIG. 2). The gm-hra probe also hybridizes to other bandscorresponding to endogenous sequences. Four plants were chosen forinsert and border sequence analysis: 3560.4.3.5 soybean plants T17 andT18, and control Jack plants C1 and C2.

In the initial characterization of the 3560.4.3.5 soybean line, theflanking genomic border regions were cloned and sequenced using theGenomeWalker and inverse PCR methods. This preliminary sequenceinformation was used to design primers for PCR to generate products inthe three regions of interest from 3560.4.3.5 soybean genomic DNA: 5′flanking border, entire insert, and 3′ flanking border (Table 11, Table4; Table 10; and, FIG. 3). Using information from the flanking bordersequence, PCR was performed on 3560.4.3.5 soybean genomic DNA andunmodified control genomic DNA.

For the 5′ flanking sequence, PCR was performed with a forward primer inthe 5′ border (primer 1679) and a reverse primer in the SCP 1 Promoter(primer 1658), resulting in the expected products in 3560.4.3.5 soybeanplants (396 bp), and not in the control DNAs (FIG. 3 and data notshown). Following two more rounds of walking (with the DNA WalkingSpeedUp and GenomeWalker Kits), additional 5′ flanking border sequencewas obtained. To verify the 5′ flanking border sequence, PCR productswere generated from 3560.4.3.5 DNA, cloned and sequenced. The completesequence information is presented in FIG. 4A-E.

For the 3′ flanking sequence, PCR was performed with a forward primer inthe gm-hra (primer 1439) and a reverse primer in the 3′ border sequence(primer 1666), resulting in the expected products in 3560.4.3.5 soybeanplants (1029 bp) and not the control DNAs (FIG. 3 and data not shown).The 3′ flanking border PCR products were generated from 3560.4.3.5soybean DNA, cloned and sequenced. This sequence showed the sameidentity to a proprietary soybean DNA sequence, which was used to designprimers to further extend the flanking border. Again, the resulting 3′flanking border PCR products generated from 3560.4.3.5 soybean DNA werecloned and sequenced. The complete sequence information is presented inFIG. 4A-E.

For amplification of the insert, PCR primers situated in each flankingregion were used to amplify the entire insert from DNA of event3560.4.3.5 plants (primer pair: 1297/1298). As expected, the predictedPCR products of 5.5 kb were generated only from 3560.4.3.5 soybean DNA,and not from the control DNA. The insert PCR products were cloned andsequenced. The sequence information is presented in FIG. 4A-E.

In addition, the DNA fragment PHP20163A used for creating 3560.4.3.5soybean line was sequenced, and compared with the insert sequencegenerated from 3560.4.3.5 soybean genomic DNA. The sequence of theinsert in 3560.4.3.5 soybean is identical to the sequence of thePHP20163A DNA fragment.

In total, 10849 bp of sequence from genomic DNA of 3560.4.3.5 soybeanwas confirmed: 3317 bp of the 5′ flanking genomic border, 2170 bp of the3′ flanking genomic border, and the 5362 bp comprising the inserted DNA(FIG. 3 and FIG. 4A-E).

To demonstrate that the identified 5′ and 3′ flanking border sequencesare of soybean origin, PCR was performed within the 5′ and 3′ flankinggenomic regions (primer pairs 1679/1514 and 1660/1666, respectively)from 3560.4.3.5 soybean DNA samples and unmodified control samples. Theexpected size PCR products (246 bp for 5′ genomic region and 297 bp for3′ genomic region) were generated using genomic DNA from both 3560.4.3.5soybean and control soybean plants, indicating that the sequences wereof soybean genomic origin and not specific to 3560.4.3.5 soybean (FIG. 3and data not data shown). These PCR products from both the 3560.4.3.5and control soybean DNAs were cloned and sequenced and shown to haveidentical sequence.

When the 3317 bp sequence from the 5′ flanking region of 3560.4.3.5soybean was compared to the Genbank Nucleotide (“nt”) dataset(www.ncbi.nlm.nih.gov/), no significant alignments were returned.Analysis using the GSS subset returned a two areas of the flankingregion (nt 2723-2863; 2881-3203) with significant similarity (98% and92%, respectively) to genomic sequences from the legume Medicagotruncatula (barrel medic). The BLASTn search against Pioneer proprietarysoybean genomic and EST sequences returned a 98% identity alignmentencompassing nt 2860-3317 to a single soybean genomic sequence. Anadditional region (nt 531-2144) returned lower, yet significantidentities (84%-92%) to several different soybean genomic clones. The 3′flanking sequence produced two highly significant alignments (97-99%identity) to two different public soybean genomic sequences (accessionsCL868338.1 and CL867466.1) and a single proprietary genomic sequence inthe region from nt 9772-10849. An additional region (nt 9250-9554)displayed significant identity (92-94%) to wheat genomic andmitochondrial sequences. The 5′ and 3′ junction regions between thesoybean genomic border sequence and the insertion in 3560.4.3.5 soybeanwere analyzed for the presence of novel open reading frames. No openreading frames greater than or equal to 100 amino acids were identifiedin the 5′ or 3′ border junction regions, indicating that no novel openreading frames were generated as a result of the insertion.

Southern blot analysis also confirmed that the DNA insertion remainedstable during traditional soybean breeding procedures. The analysis wasconducted on two self-crossed generations, T4 and T5, and verified thatthe insertion remained intact and stably integrated as demonstrated byidentical hybridization patterns in the two generations. The F3generation was also analyzed by Southern blot analysis and confirmed thesame stable, event-specific hybridization pattern as exhibited by the T4and T5 generations. These results confirmed the stability of theinsertion in 3560.4.3.5 soybean across multiple breeding generations.

As discussed below, the Bgl II restriction enzyme has a single site (bpposition 2254) located within the PHP20163A fragment (FIG. 6) and willgenerate a unique event-specific hybridization pattern for 3560.4.3.5soybean when hybridized to the glyat4601 and gm-hra probes. Thisanalysis confirms event stability across generations as changes to theinsertion structure in 3560.4.3.5 soybean would be detected. Asdiscussed below, a band of approximately 2500 bp would be expected withthe glyat4601 probe to confirm stability across generations (Table 5).Likewise, for the gm-hra probe, a band of approximately 3500 bp would beexpected to confirm stability across generations (Table 6).

TABLE 5 Predicted and Observed Hybridizing Bands on Southern Blots withglyat4601 Cassette Probes Observed Predicted Fragment Size PredictedFragment Size from in GLYAT Restriction Fragment Size from PHP20163²3560.4.3.5 Probe Enzyme PHP20163A¹ (bp) (bp) soybean³ (bp) SCP1 promoterBgl II >2300⁴ 3485  ~2500 glyat4601 Bgl II >2300⁴ 3485  ~2500 pinIIterminator Bgl II >2300⁴ 3485  ~2500 SCP1 promoter Xba I  1379 1379⁵ 1379⁶ glyat4601 Xba I  1379 1379⁵  1379⁶ pinII terminator Xba I  13791379⁵  1379⁶ ¹Predicted fragment sizes for 3560.4.3.5 soybean are basedon the map of PHP20163A as shown in FIG. 6. ²Predicted fragment sizesfor hybridization to samples containing the plasmid positive control arebased on the PHP20163 plasmid map as shown in FIG. 7. ³Observed fragmentsizes are considered approximate from these analyses and are based onthe indicated sizes of the DIG-labeled DNA Molecular Weight Marker VIIfragments on the Southern blots. Due to incorporation of DIG moleculesfor visualization, the marker fragments typically run approximately5-10% larger than their actual indicated molecular weight. ⁴Minimumfragment size predicted based on an intact insertion of PHP20163A (datanot shown). ⁵Predicted hybridizing Xba I fragment size from plasmidPHP20163 grown in a Dam⁻ strain. For plasmid grown in a Dam⁺ strain, thehybridizing Xba I fragment size is predicted to be 5307 bp. ⁶Observedfragment is equal to the predicted size based on blot (data not shown)showing comparison to plasmid PHP20163 grown in a Dam⁻ strain.

TABLE 6 Predicted and Observed Hybridizing Bands on Southern Blots withgm-hra Cassette Probes

An asterisk (*) and gray shading indicates the designated band is due toprobe hybridization to endogenous soybean genome sequences, as can bedetermined by the presence of the same band in all lanes, both3560.4.3.5 soybean and control. ¹Predicted fragment sizes for 3560.4.3.5soybean are based on the map of PHP20163A as shown in FIG. 6. ²Predictedfragment sizes for hybridization in samples containing the plasmidpositive control are based on the PHP20163 plasmid map as shown in FIG.7. ³Observed fragment sizes are considered approximate from theseanalyses and are based on the indicated sizes of the DIG-labeled DNAMolecular Weight Marker VII fragments on the Southern blots. Due toincorporation of DIG molecules for visualization, the marker fragmentstypically run approximately 5-10% larger than their actual indicatedmolecular weight. ⁴Minimum fragment size predicted based on an intactinsertion of PHP20163A (data not shown). ⁵Predicted hybridizing Xba Ifragment size from plasmid PHP20163 grown in a Dam strain. For plasmidgrown in a Dam⁺ strain, the hybridizing Xba I fragment size is predictedto be 5307 bp. ⁶Observed fragment is equal to the predicted size basedon blot (data not shown) showing comparison to plasmid PHP20163 grown ina Dam⁻ strain.

Genomic DNA of T4 and T5 generations of 3560.4.3.5 soybean was digestedwith Bgl II and hybridized to the glyat4601 and gm-hra probes to confirmstability across generations (data not shown). A band of approximately2500 bp specific to 3560.4.3.5 soybean hybridized to the glyat4601 probein both the T4 and T5 generations (Table 5 and data not shown). With thegm-hra probe, a single band of approximately 3500 bp specific to3560.4.3.5 soybean was present in both generations (Table 6 and data notshown). In addition to the 3500 bp band, the gm-hra probe alsohybridized to additional bands that were determined to be endogenous tothe soybean genome since these bands were present in both 3560.4.3.5soybean and control soybean plants (data not shown). Hybridizationresults from both the glyat4601 and gm-hra probes confirmed that theinsertion of the PHP20163A DNA fragment in 3560.4.3.5 soybean remainedstable across the self-crossed T4 and T5 generations.

Southern blot analysis of the F3 generation of 3560.4.3.5 soybean wasalso conducted. A total of 77 individual plants were analyzed (data notshown). Genomic DNA of the F3 generation was digested with Bgl II andhybridized to the glyat4601 and gm-hra probes. A band of approximately2500 bp was observed with the glyat4601 probe (data not shown) and asingle band of approximately 3500 bp specific to 3560.4.3.5 soybean wasobserved with the gm-hra probe (data not shown). As with the previousanalysis conducted, the gm-hra probe hybridized to additional bands in3560.4.3.5 soybean and control samples which were due to endogenoussequences within the soybean genome (data not shown). Hybridizationsresults from both the glyat4601 and gm-hra probes were consistent withthe results from the T4 and T5 generations described above and confirmedthe stability of inheritance of the insertion during traditional soybeanbreeding.

Both the T4 and T5 generations were analyzed to confirm the absence ofplasmid sequence from plasmid PHP20163 outside of the transformationfragment PHP20163A, i.e. the plasmid backbone sequence removed prior totransformation. The results verified the absence of backbone sequencesin 3560.4.3.5 soybean.

The backbone 20163 and hyg 20163 probes (Table 3 and FIG. 7) weredesigned to hybridize to areas of plasmid PHP20163 outside of thetransformation fragment (data not shown) and were hybridized to XbaI-digested genomic DNA to confirm absence of these sections of theplasmid in 3560.4.3.5 soybean. Neither of the two backbone probeshybridized to 3560.4.3.5 soybean samples (data not shown), confirmingthe absence of these sequences.

Genomic DNA from leaf material of the Jack soybean variety was used as anegative control for all Southern blot analyses. Genomic DNA from anelite soybean variety (Elite 1) was included as an additional negativecontrol for analysis of the F3 generation. Plasmid PHP20163 was used asa positive control for probe hybridization and to verify fragment sizesinternal to the transformation fragment PHP20163A. All probes used forthe analysis are indicated on the schematic maps of PHP20163A andPHP20163 (FIGS. 6 and 7, respectively) and outlined in Table 3.

The integration pattern of the insertion in 3560.4.3.5 soybean wasinvestigated with Bgl II digestion to determine copy number and with XbaI digestion to determine insertion integrity. Southern blots werehybridized to several probes to confirm copy number and integrity ofeach genetic element. SCP1 promoter, glyat4601, and pinII terminatorprobes were used to characterize the glyat4601 cassette (Table 3 datanot shown). SAMS, gm-hra, and als terminator probes were used tocharacterize the gm-hra cassette (Table 3 and data not shown).

The Bgl II digest provides information about number of copies integratedinto the genome of 3560.4.3.5 soybean as there is a single restrictionenzyme site in the PHP20163A fragment at base pair (bp) position 2254(data not shown) and additional sites outside the fragment in thesoybean genome. Hybridization with the probes from each cassette, exceptfor the SAMS probe, would indicate the number of copies of each elementfound in 3560.4.3.5 soybean based on the number of hybridizing bands(e.g. one hybridizing band indicates one copy of the element). For theSAMS probe, since the Bgl II site is located within the probe region,two hybridizing bands would be expected for every one copy of theelement. Predicted and observed fragment sizes for 3560.4.3.5 soybeanwith Bgl II are given in Table 5 for the glyat4601 cassette and in Table6 for the gm-hra cassette.

Based on the Southern blot analyses as discussed below, it wasdetermined that a single, intact PHP20163A fragment has been insertedinto the genome of 3560.4.3.5 soybean as diagramed in the insertion map(FIG. 8).

A single copy of all the elements of the glyat4601 cassette was insertedinto 3560.4.3.5 soybean. SCP1 promoter, glyat4601, and pinII terminatorprobes were hybridized to Bgl II-digested genomic DNA from individual3560.4.3.5 soybean plants of the T4 generation (Table 5 and data notshown). Each of the probes hybridized to the same single-fragment ofapproximately 2500 base pairs (bp) (Table 5 and data not shown),indicating the expected arrangement of genetic elements on the fragmentinserted in 3560.4.3.5 soybean.

Likewise, a single copy of all the elements of the gm-hra cassette wasinserted into 3560.4.3.5 soybean. The elements comprising thiscassette—the SAMS promoter region, gm-hra gene, and als terminator—wereused as probes to determine number of copies inserted. The probes ofthis cassette are homologous to elements endogenous to the soybeangenome and therefore each probe hybridized to bands in control soybeansamples. The hybridizing bands in 3560.4.3.5 soybean from the endogenoussoybean genome are indicated by asterisks in the shaded boxes of Table 6and were determined by their presence in control soybean samples and arethus not associated with the insertion.

The SAMS probe hybridized to one band of approximately 2500 bp and asecond band of approximately 3500 bp in 3560.4.3.5 soybean (Table 6 anddata not shown, SAMS probe). Two bands would be expected to indicate oneinsertion of this element with this probe as the Bgl II site is locatedwithin the SAMS region of PHP20163A (FIG. 6) and the results indicatedone copy of the element. The 2500 bp fragment was determined to be thesame fragment containing the glyat4601 cassette as described above.

The gm-hra and a/s terminator probes hybridized to the same 3500 bpfragment as the SAMS probe (Table 6 and data not shown, gm-hra andgm-als terminator probes). The hybridization of all three probes to thesame 3500 bp fragment and of the SAMS probe to the 2500 bp fragmentconfirmed the expected arrangement of the genetic elements in the DNAinsertion in 3560.4.3.5 soybean.

Xba I digestion was used to verify that the glyat4601 and gm-hracassettes were complete and intact in 3560.4.3.5 soybean as there arethree sites in the PHP20163A fragment (base pair positions 8, 1387, and5315) which precisely flank each gene expression cassette (FIG. 6).Hybridization with the probes of the glyat4601 and gm-hra cassettesconfirmed that all the elements were found on the appropriate internalfragments containing the cassette. Expected and observed fragment sizeswith Xba I are given in Table 6 for the glyat4601 cassette and Table 6for the gm-hra cassette.

The SCP1 promoter, glyat4601, and pinII terminator probes eachhybridized to the expected internal band of 1378 bp (Table 5 and datanot shown) and the size was confirmed by additional hybridizations asdescribed in the section below and data not shown. Because these probeshybridized to the same internal fragment of the predicted size, theglyat4601 cassette in 3560.4.3.5 soybean was determined to be intact andall elements of the cassette were confirmed on this fragment.

The SAMS, gm-hra, and als terminator probes each hybridized to theexpected internal band of 3927 bp band (Table 6 and data not shown) andthe size was confirmed by additional hybridization described in thesection below. Because these probes hybridized to the same fragment, thegm-hra cassette in 3560.4.3.5 soybean was determined to be intact andall elements were present.

Plasmid PHP20163 was prepared from a strain of E. coli that expresses aDNA methylase (a Dam⁺ strain). This plasmid did not produce the expectedbands when digested with Xba I and probed with the glyat4601 and gm-hracassette probes. A band of approximately 5300 bp was observed in lanescontaining plasmid PHP20163 for all probes (data not shown) instead ofthe predicted 1379 bp and 3928 bp size bands (Tables 5 and 6). Based onthe plasmid sequence, it was determined that the Xba I site at bpposition 1387 of PHP20163 (FIG. 7) overlaps a Dam methylationrecognition sequence. The final adenine in this site is methylated, thusblocking digestion by Xba I.

The inability for Xba I enzyme to cut at this site affected the sizeprediction from fragment PHP20163A in 3560.4.3.5 soybean (Tables 5 and6, FIG. 6). In order to confirm the size in 3560.4.3.5 soybean and theplasmid result, plasmid PHP20163 was prepared from a strain of E. colilacking Dam methylase (Dam⁻ strain) and was compared to the plasmidPHP20163 (data not shown). Southern hybridization results (data notshown) show the plasmid comparison alongside samples of the T4 and T5generations of 3560.4.3.5 soybean digested with Xba I. The blot probedwith glyat4601 and gm-hra probes demonstrated that plasmid PHP20163prepared from the Dam⁻ strain digested as expected by Xba I and producedthe predicted size bands of 1379 bp and 3927 bp (data not shown), whilethe original plasmid from the Dam⁺ strain again produced a band ofapproximately 5300 bp for both probes (data not shown). These resultsconfirmed that the central Xba I site at position 1387 was blocked fromdigestion due to Dam methylation. Furthermore, the hybridizing bands in3560.4.3.5 soybean were of the equivalent size as those in theunmethylated plasmid PHP20163 from the Dam⁻ strain confirming that3560.4.3.5 soybean contained a complete and intact insertion (data notshown).

A Chi squared analysis of trait inheritance data from five differentgenerations (T1, F2, F3, BC1F2, C2F2) was performed to determine theMendelian heritability and stability of the glyat4601 and gm-hra genesin the progeny of 3560.4.3.5 soybean. The breeding history of the fivegenerations evaluated for Mendelian inheritance is described in FIG. 5.For each of the generations tested, the plants were expected tosegregate 1:2:1 (homozygous positive plants:hemizygous plants:homozygousnegative [null] plants). Various approaches, as outlined below, wereused to confirm this segregation ratio in the progeny of 3560.4.3.5soybean.

In some studies, homozygous positive plants were not differentiated fromhemizygous plants, resulting in a 3:1 positive:negative segregationpattern (Table 7). For the three generations listed in Table 7, threedifferent methods, respectively, were used to score the plants aspositive or negative:

-   -   qualitative PCR analysis to identify the plants containing the        glyat4601 gene (T1 generation); or    -   western analysis to score plants for GLYAT4601 protein        expression followed by confirmation of those same plants by        Southern analysis with both glyat4601 and gm-hra probes (F3        generation), or    -   an ALS seed soak assay (BC1F2 generation). In this assay, seeds        are soaked in an ALS-inhibiting herbicide containing the active        ingredient chlorsulfuron. Only seed expressing the gm-hra gene        will emerge after planting.

In certain studies, plants that did not contain either the glyat4601 orgm-hra genes were removed from the study prior to the conduct ofsegregation analysis. Remaining plants were then scored as homozygouspositive or hemizygous, resulting in a 1:2 homozygouspositive:hemizygous segregation pattern (Table 8).

For the F2 generation listed in Table 8, two methods were used to removethe negative plants and score the remaining plants as homozygouspositive or hemizygous:

-   -   A glyphosate spray was, applied after the plants had emerged,        removing all of the homozygous negative (null) plants. This was        followed by an ALS-inhibiting herbicide “ragdoll” assay for the        gm-hra gene, where progeny seed from the F2 plants were screened        to determine the F2 parent plant genotype. In the ragdoll assay,        paper towels were wetted with an ALS-inhibiting herbicide        containing the active ingredient chlorsulfuron. Ten progeny        seeds from a single F2 parent plant were rolled into the wetted        towel and allowed to germinate. An F2 parent plant was scored as        homozygous positive for the gm-hra gene if all ten progeny seeds        germinated and grew normally. An F2 parent plant was scored as        homozygous negative for the gm-hra gene if all ten progeny seeds        did not germinate and grow normally. A hemizygous F2 parent        genotype was characterized by having a mixture of resistant and        susceptible plants within the ten seed sample.    -   An ALS seed soak assay of the seeds prior to planting removed        all of the homozygous negative (null) plants, followed by a        quantitative real time PCR (qPCR) assay to distinguish plants        that were homozygous positive or hemizygous for the glyat4601        gene.

In some studies, all plants were identified as homozygous positive,hemizygous, or homozygous negative to confirm a 1:2:1 expectedsegregation ratio (Table 9). Segregation analysis was conducted for theC2F2 generation using quantitative real time PCR (qPCR) assays for boththe glyat4601 and gm-hra genes. Because the glyat4601 and gm-hra geneswere physically linked in the DNA fragment used for transformation andare expected to have identical segregation ratios in the progeny of3560.4.3.5 soybean, the glyat4601 results are applicable to theinheritance of gm-hra and vice versa. In generations where both traitswere analyzed in the same plants (F3 and C2F2), identical segregationdata would experimentally confirm co-segregation of the glyat4601 andgm-hra genes

Results from the Mendelian analysis are summarized in Tables 7, 8 and 9.All P-values were greater than 0.05, indicating no statisticallysignificant differences between the observed and expected frequencies ofthe glyat4601 and/or gm-hra genes in five generations of 3560.4.3.5soybean. The results of this analysis are consistent with the finding ofa single locus of insertion of the glyat4601 and gm-hra genes thatsegregates in 3560.4.3.5 soybean progeny according to Mendel's laws ofgenetics. The stability of the insert has been demonstrated in fivegenerations of self- and cross-pollinations.

TABLE 7 Comparison of Observed and Expected 3:1 Segregation Ratios for3560.4.3.5 Soybean Observed Expected Chi-squared Positives NegativesPositives Negatives test Generation Method +/+ or +/− −/− +/+ or +/− −/−P-value T1 Glyat4601 PCR 59 23 61.5 20.5 0.610 F3 Elite 1 GLYAT 75 1567.5 22.5 0.088 background 4601 westerns followed by Southern analyseswith glyat4601 and gm-hra probes BC1F2 ALS seed soak Elite 7 700 222691.5 230.5 0.543 background Elite 8 761 273 775.5 258.5 0.315background Elite 9 160 54 160.5 53.5 1.000 background Elite 10 205 79213 71 0.304 background

TABLE 8 Comparison of Observed and Expected 1:2 Segregation Ratios for3560.4.3.5 Soybean Observed Expected Chi-squared Homozygous HemizygousHomozygous Hemizygous test Generation Method +/+ +/− +/+ +/− P-value F2Elite 1 Glyphosate spray 16 24 13.3 26.7 0.467 background to removenulls Elite 2 and followed by 32 53 28.3 56.7 0.466 background ALSinhibitor ragdoll test Elite 3 ALS seed soak to 110 182 97.3 194.7 0.131background remove nulls Elite 4 followed by 124 284 136 272 0.227background qPCR for Elite 5 glyat4601 27 61 29.3 58.7 0.678 backgroundElite 6 22 29 17 34 0.181 background

TABLE 9 Comparison of Observed and Expected 1:2:1 Segregation Ratios for3560.4.3.5 Soybean Chi- squared Gen- Observed Expected test erationMethod +/+ +/− −/− +/+ +/− −/− P-value C2F2 Elite 44 Glyat4601 41 76 4340 80 40 0.799 back- and gm- ground Elite 45 hra qPCR 160 294 142 149298 149 0.550 back- ground

TABLE 10 Description and Sizes of PCR Products Forward Reverse Size (bp)of Primer Primer Description of amplified region PCR product 1297 1298Inserted DNA 5457 1679 1658 5′ flanking genomic border to SCP1 396Promoter 1679 1514 5′ flanking genomic border 246 1439 1666 gm-hra to 3′flanking genomic border 1029 1660 1666 3′ flanking genomic border 297

TABLE 11 List of Primer Sequences Used in PCR Reactions TargetAbbreviated Sequence SEQ Primer Primer Location ID Name NameSequence (5′-3′) (bp to bp)¹ NO 05-0-1227 1227TGGTCTTCTGAGACTGTATCTTTGATATTC  3402-3373 24 05-0-1297 1297TGCCCGAGGTCGTTAGGTCGAATAGGCTAG  3268-3297 25 05-0-1298 1298TCCTATTCAAGATGGGCAGTGTCTTCCTAATGATG  8724-8690 17 06-0-1439 1439GATAACTGAGGGTGATGGTAGAACGAGGTACTGATTG  7951-7987 18 06-0-1440 1440TGTGATAACTGAGGGTGATGGTAGAACGAGGTACTG  7948-7983 26 06-0-1473 1473TTATTTCCGATCGGATCCTGCCAGTGGAG 10849-10821 50 06-0-1504 1504CCACCATGTTGACGGATCTCTAG  3347-3325 51 06-0-1505 1505GCAATGGAATCCGAGGAGGTTTC  3463-3441 52 06-0-1506 1506GCAATGATGGCATTTGTAGGTG  3532-3511 53 06-0-1514 1514TCGATCGGTCAAGAATCCGGTTCTC  3263-3239 19 06-0-1549 1549AAACTGAAGCGATGGCAGAACCGCACAG  1962-1935 54 06-0-1550 1550TCGAAGTGGCAATAGAGCCACACAATATCGATAAG  2012-1978 55 06-0-1610 1610AGCAATTGTTTTGTGCATTTCCAAATTTCAATCTG     1-35 40 06-0-1658 1658CAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTG  3413-3379 20 06-0-1660 1660GTAATATCATCATTAGGAAGACACTGCCCATCTTG  8683-8717 21 06-0-1666 1666CCATATTTGAAAGCCTAAGCAGATGGCATAATTC  8979-8946 22 06-0-1679 1679TTCAGCAACAAACTCTCATCGTGAGCAG  3018-3045 23 ^(1.)Location in sequence of346043 soybean. (See FIG. 4) Bases 1-3317 = 5′ genomic border, bases3318-8679 = insert, basese 8680-10849 = 3′ genomic border.

TABLE 12 Additional Primers for Detecting 3560.4.3.5 soybean SEQ IDPrimer Characteristics NO GGTCGAATAGGCTAGGTTTAC(start 751, length 24 nt, 7 GAA tm 59, % GC 59) AAGAGACTAAGGCCGCTC(taqman mgb probe, start 9 776, length 18, tm 68, % gc 56)CCACCATGTTGACGGATCTCT (start 815, length 21, 8 tm 58)GTCGAATAGGCTAGGTTTACG primer DP-356-f1 37 AAAAA TTTGATATTCTTGGAGTAGACPrimer Dp-356-r1 38 GAGAGTGT 6FAM-CTCTAGAGATCCGTCADP356-p (Taqman probe) 39 ACATGGTGGAGCAC-TAMRA

Example 2 Additional methods to identify a 3560.4.3.5 Event

Oligonucleotide PCR Reagents:

Forward Primer: 5′ GGTCGAATAGGCTAGGTTTACGAA (SEQ ID NO: 7)Reverse Primer: 5′ CCACCATGTTGACGGATCTCT (SEQ ID NO: 8)Taqman MGB probe: 5′ Fam-AAGAGACTAAGGCCGCTC-MGB 3′ (SEQ ID NO: 9)Each primer is used at a concentration of 900 nM in the PCR. The MGBprobe was used at a concentration of 80 nM in the PCR. The PCR mixtureused was “Extract-N-Amp. PCR Ready Mix” (Cat. No. E3004) fromSigma-Aldrich. Rox reference dye was also included in the PCR mixture byadding 0.01 volumes of Sigma-Aldrich “Reference Dye for QuantitativePCR” (Cat. No. R4526). PCR was performed for 40 cycles with one cycleconsisting of the following two steps: Step 1: 15 seconds at 95° C. andStep 2: 60 seconds at 60° C. Amplicon product had a size of 85 bp.

Additional primers that can be used to identify a 3560.4.3.5 soybeanevent are set forth in Table 13.

TABLE 13 SEQ Product ID Primer Sequence Size (bp) NO 104312AGATCCGTCAACATGGTGGAGCAC 44 104314 TGACAGATAGCTGGGCAATGGAATCC 150 45Probe 125323 6FAM-TATCGGGAAACCTC-MGB 46 109893CTTTGCTGTTTGATTGCTGGGTTGTC 47 (endogenous control) 109894TGTGTGGACCCATTGGCCTTTAGATTAT 144 48 (endogenous control) ProbeVIC-ACTCTGCAGTTGCCTT-MGB 49 125322 (endogenous control)These primers were used in a presence/absence assay to detect a3560.4.3.5 soybean event. Primers 104312+104314/SCPITP10 detect thetransgenic insertion; control primers, such as, 109893+109894/probeP94032A2 were used to detect house-keeping gene (aspartateaminotransferase gene) and were used as internal control. A 3560.4.3.5soybean event will show heterozygous signals (FAM and VIC) and thenon-transgenic genotypes will show homozygous P94032A2 (VIC) positive.PCR conditions employed in this method of detecting the 3560.4.3.5soybean are shown below.

TABLE 14 PCR Conditions Initial denat 120 sec  95 C. Anneal 90 sec 66 C.Extend 90 sec 72 C. Denat 30 sec 95 C. 14 cycles Final extend 120 72 C.Initial denat 120 sec  95 C. Anneal 60 sec 60 C. Extend 10 sec 82 C.Denat 30 sec 95 C. Final extend  0 82 C. 32 cycles

Those skilled in the art would also include a control PCR using anendogenous gene to verify that the isolated genomic DNA was suitable forPCR amplification. Soybean endogenous genes that have been usedsuccessfully with soybean samples are the following: Lectin gene(Schmidt, M and Parrott, W, 2001) and conglycinin α′-subunit gene(Shirai (1998) Biosci Biotechnol Biochem 62:1461-1464). Another locationto find endogenous gene targets for PCR is the web site(www.//biotechjrc.it) which is sponsored by the Joint Research Centre(JRC) of the European Commission. See, also, Schmidt et al. (2001) PlantCell Rep 20:422-428.

Example 3

Soybean event 3560.4.3.5 was selected as a lead event and BC1F3 lineswere created by backcrossing 3560.4.3.5 soybean into four differentconventional lines. Across these four backcross populations, lines withthe 3560.4.3.5 event (sprayed with glyphosate) were not significantlydifferent for yield compared to negative null segregant lines (sprayedwith conventional herbicides). Soybean event 3560.4.3.5 confers a highlevel of tolerance to glyphosate and ALS inhibitor herbicides with noyield impact.

The first objective of this study was to evaluate 3560.4.3.5 soybean todetermine the level of tolerance to different application rates ofglyphosate and ALS inhibitor herbicides. The second objective was todevelop homozygous positive and homozygous negative null segregants(NILs) of 3560.4.3.5 soybean event to determine if yield was impacteddue to presence of the 3560.4.3.5 event. The third objective was tointegrate event 3560.4.3.5 into different genetic backgrounds todetermine if there was any impact on herbicide tolerance or yieldperformance.

Materials and Methods

For early generation transgenic event testing, TO plants were sprayed atthe V2 to V6 growth stage (Fehr et al. (1977) Coop. Ext. Ser. SpecialReport 80. Iowa State Univ., Ames Iowa) with 1.68 kg ae ha⁻¹ or 3.36 kgae ha⁻¹ glyphosate plus adjuvants (0.25% v/v nonionic surfactant(NIS)+2.24 kg ai ha⁻¹ ammonium sulfate (AMS)) in the Newark, Del.greenhouses. Plants were rated using a 1 to 9 scale ten days aftertreatment (DAT), where 1=dead plant to 9=no observed spray response.

Selected events were advanced at the T1 generation for herbicidetolerance confirmation in greenhouses. Plants were sprayed at the V3growth stage with either 1.68 kg ae ha⁻¹ glyphosate, 60 g ai ha⁻¹thifensulfuron methyl (methyl3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate),or a sequential application of 70 g ai ha⁻¹ thifensulfuron methyl at V3followed by 2.24 kg ai ha-1 glyphosate at V6. All spray treatmentsincluded 0.25% v/v NIS+2.24 kg ai ha⁻¹ AMS. At ten days after sprayapplication, plants were rated using a 1 to 9 scale. Since the T1 eventswere segregating, only the non-susceptible plants were scored, and anaverage rating of tolerant plants within each event was calculated.

Plants from individual T1 events were grown separately in 2.4 m rows(76.2 cm row spacing). Presence or absence of glyat+hra in eachindividual T1 plant was determined by using polymerase chain reaction(PCR) amplification of the glyat4601 insert. PCR results for each eventwere analyzed using chi square analyses to identify events withMendelian segregation. For each of the events evaluated, T1 plants thatwere positive for the glyat+hra were harvested separately and advancedas plant-to-row T2 short rows (2.4 m rows with 76.2 cm row spacing).Twelve remnant seed from each T2field entry were grown and V3 plantswere sprayed with 3.36 kg ae ha¹ glyphosate plus 0.25% v/v NIS+2.24 kgai ha⁻¹ AMS to determine zygosity of the corresponding short row. Lineswere considered homozygous positive if all 12 plants were tolerant,homozygous negative if all 12 plants were susceptible, and heterozygousif the 12 plants were a mixture of tolerant and susceptible phenotypes.

Chi square was used to analyze T2 results to identify events withMendelian segregation across generations. Twenty four different lines atthe T3 generation with the glyat+hra were selected for advancement toherbicide tolerance trials. Homozygous positive and homozygous negativeNILs from ten events were advanced to isoline yield trials. 3560.4.3.5soybean event was one of the events selected for advancement.

The 3560.4.3.5 soybean event was evaluated for glyphosate tolerance.3560.4.3.5 soybean was grown in a randomized complete block (RCB) designin a split plot arrangement with three replications of paired 3.7 m rows(76.2 cm row spacing). Treatments consisted of an unsprayed control, andthree spray rates applied at the V5 growth stage using either 3.36 kg aeha⁻¹, 6.72 kg ae ha⁻¹, or 13.44 kg ae ha⁻¹ glyphosate. Plots wereevaluated for crop response at 7 and 14 days after treatment (DAT), andrated using a 1-9 score. Crop response data were analyzed using thegeneral linear model (GLM) and mixed model analysis of variance (ANOVA)procedures of SAS. (The SAS System is a registered trademark of SASInstitute, Inc., Cary, N.C., USA)

Selected T4 lines were tested for glyphosate tolerance. This experimentwas a RCB design in a split plot arrangement with two replications ofindividual 1.2 m rows (76.2 cm row spacing). At the V3 growth stage,6.72 kg ae ha⁻¹ glyphosate was applied to one block, while another wasunsprayed for use as a control. The third block received a sequentialapplication of 6.72 kg ae ha⁻¹ glyphosate at V3 followed by 6.72 kg aeha⁻¹ glyphosate at R1. Plots were evaluated for crop response at 7 DATand 14 DAT, and assigned a visual response score from 0% to 100%response, where 0%=no response observed to 100%=plot completely dead.Crop response data were analyzed using the GLM and mixed model ANOVAprocedures of SAS.

ALS inhibitor herbicide tolerance of the gm-hra gene was compared tosulfonylurea tolerant soybeans (STS®) by growing T4 plants of event3560.4.3.5 and a STS® cultivar. The experiment was designed as a RCBdesign in a split plot arrangement with two replications of paired 3.7 mrows (76.2 cm row spacing). Spray treatments were applied at the V3growth stage and consisted of one of the following: an unsprayedcontrol, 8.8 g ai ha⁻¹ rimsulfuron(N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-3-(ethylsulfonyl)-2-pyridinesulfonamide),8.8 g ai ha⁻¹ tribenuron methyl (methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]amino]sulfonyl]benzoate),or 30.0 g ai ha⁻¹ chlorimuron ethyl (ethyl2-[[[[(4-chloro-6-methoxypyrimidin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate)+9.67g ai ha⁻¹ thifensulfuron methyl. All spray treatments listed had 0.25%v/v NIS+2.24 kg ai ha⁻¹ AMS added. Plots were evaluated for cropresponse at 7 DAT and 14 DAT, and assigned a visual response score from0% to 100% response. Crop response data were analyzed using the mixedmodel ANOVA procedure of SAS.

ALS inhibitor herbicide tolerance trials were completed at two separatelocations. Plants from the T5 generation of event 3560.4.3.5 and a STS®cultivar were grown in a RCB design in a split plot arrangement withthree replications of paired 2.4 m rows (76.2 cm row spacing).Treatments were applied at the V3 growth stage and consisted of anunsprayed control, 4.2 g ai ha⁻¹ metsulfuron methyl (methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate),70.0 g ai ha⁻¹ thifensulfuron methyl, and 17.5 g ai ha⁻¹ tribenuronmethyl. All spray treatments listed had 0.25% v/v NIS+2.24 kg ai ha⁻¹AMS added. Visual response ratings (0% to 100%) were assigned to eachplot at 7 DAT, 14 DAT, and 28 DAT. Crop response data were analyzedusing the mixed model ANOVA procedure of SAS.

Multiple trials were conducted with the lead event 3560.4.3.5 soybean toobserve the level of tolerance to different rates and applicationtimings of glyphosate with and without other pesticide mixtures. Thefirst experiments were grown in an RCB design of three replications ofpaired 3.7 m rows (76.2 cm row spacing), blocked by replication withrandomized spray treatments within each replication. Treatments wereapplied at the V2 or R2 growth stage, and consisted of the following:unsprayed control, 3.36 kg ae ha⁻¹ glyphosate, 3.36 kg ae ha⁻¹glyphosate+560.4 g ai ha⁻¹ chlorpyrifos (O,O-diethylO-3,5,6-trichloro-2-pyridyl phosphorothioate), 3.36 kg ae ha⁻¹glyphosate+1120.9 g ai ha⁻¹ bentazon(3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide), 3.36 kg aeha⁻¹ glyphosate+141.3 g ai ha⁻¹ imazethapyr(2-[4.5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylicacid), 3.36 kg ae ha⁻¹ glyphosate+263.4 g ai ha⁻¹ fomesafen(5-[2-chloro-4-(trifluoromethyl)phenoxy]-N-(methylsulfonyl)-2-nitrobenzamide),3.36 kg ae ha⁻¹ glyphosate+17.5 g ai ha⁻¹ thifensulfuron methyl+17.5 gai ha⁻¹ tribenuron methyl, 3.36 kg ae ha⁻¹ glyphosate+17.5 g ai ha⁻¹thifensulfuron methyl+17.5 g ai ha⁻¹ tribenuron methyl+560.4 g ai ha⁻¹chlorpyrifos, 3.36 kg ae ha⁻¹ glyphosate+17.5 g ai ha⁻¹ thifensulfuronmethyl+17.5 g ai ha⁻¹ tribenuron methyl+1120.9 g ai ha⁻¹ bentazon, 3.36kg ae ha⁻¹ glyphosate+17.5 g ai ha⁻¹ thifensulfuron methyl+17.5 g aiha⁻¹ tribenuron methyl+141.3 g ai ha⁻¹ imazethapyr, and 3.36 kg ae ha⁻¹glyphosate+17.5 g ai ha⁻¹ thifensulfuron methyl+17.5 g ai ha⁻¹tribenuron methyl+263.4 g ai ha⁻¹ fomesafen. All spray treatments listedhad 0.25% v/v NIS+2.24 kg ai ha⁻¹ AMS added. Visual response ratings (0%to 100%) were assigned to each plot at 7 DAT, 14 DAT, and 28 DAT. Cropresponse data were analyzed using the GLM and mixed model ANOVAprocedures of SAS.

The second set of tolerance experiments were designed to measure yieldpotential of 3560.4.3.5soybean after application of common field userates of glyphosate and/or ALS inhibitor herbicides. These experimentswere six replications of paired 3.7 m rows (76.2 cm row spacing), grownin a RCB design, blocked by replication, with randomized spraytreatments within each replication. One experiment was grown at field Aand two experiments were grown at field B and C (which were separated byplanting date and physical distance of field locations.) In addition,the two fields had different environmental influence; one fieldexperienced drought stress through the spring and early summer, theother was well irrigated throughout the growing season. Treatments wererandomized within each of the replications and sprayed at the VC, V2,R2, or R5 growth stages. Treatments consisted of 1.68 kg ai ha⁻¹glyphosate, 5.8 g ai ha⁻¹ chlorimuron ethyl+4.4 g ai ha-1 thifensulfuronmethyl, and 1.68 kg ai ha⁻¹ glyphosate+5.8 g ai ha⁻¹ chlorimuronethyl+4.4 g ai ha⁻¹ thifensulfuron methyl. All spray treatments listedhad 0.25% v/v NIS+2.24 kg ai ha⁻¹ AMS) added. Visual response ratings(0% to 100%) were assigned to each plot at 7 DAT, 14 DAT, and 28 DAT.Plots were harvested and yield was calculated for each plot. Cropresponse data were analyzed using the GLM and mixed model ANOVAprocedures of SAS.

Homozygous positive and homozygous negative null segregants (NILs) of3560.4.3.5 soybean from T3 generation events were grown in preliminaryyield trials to determine if yield was impacted due to the presence of3560.4.3.5 event. Isoline yield trials were designed as a RCB (blockedby event), with a single replication of paired 3.7 m rows (76.2 cm rowspacing). Trials were mechanically cultivated and/or had labeled userates of conventional soybean herbicides applied, as needed, to ensureweed-free conditions. Maturity scores and yield data were collected foreach entry and subject to ANOVA using the mixed model procedure of SAS.

T3 seed for each isoline entry was also advanced to the T4 generation.Remnant T3 seed from each line was confirmed to be either homozygouspositive or homozygous negative by evaluating 12 seedlings that weregerminated in chlorsulfuron herbicide solution. Conventional soybeanlines are not tolerant to spray application of chlorsulfuron, which iscurrently utilized in small grain production to control broadleaf weeds.The chlorsulfuron herbicide stock solution was created by mixing 66.7 mgof chlorsulfuron with 1 liter of water. The mixture was buffered to a pHof 7.5 using 1 mM phosphate buffer. From this stock solution, 20 ml wasmixed with 1 liter of water, which was then used to saturate germinationpaper. Seeds were added to the germination paper and observed 10 daysafter initial treatment for their response. Seedlings that possessed the3560.4.3.5 event germinated and grew normally, while seedlings thatlacked the event created hooked unifoliolate leaves that did not expandand develop any further (data not shown). Lines were scored ashomozygous positive if all 12 seedlings grew normally through thesolution (data not shown). Lines were scored as homozygous negative ifall 12 plants had the inhibited hooked unifoliolate phenotype (data notshown).

Homozygous positive 3560.4.3.5 event and homozygous negative NILs weregrown in isoline yield trial experiments. Trials were mechanicallycultivated and/or had labeled use rates of conventional soybeanherbicides applied, as needed, to ensure weed-free conditions. Maturityscores and yield data were collected for each entry and subject to ANOVAusing the mixed model procedure of SAS.

Remnant T4 seed from each line was confirmed to be either homozygouspositive or homozygous negative by evaluating 12 seedlings using thechlorsulfuron herbicide screen. T4 seed for each selected isoline entrywas advanced to the T5 generation.

Event 3560.4.3.5 was selected for additional isoline yield trialevaluations. Homozygous positive and homozygous negative NILs from event3560.4.3.5 were grown using the same experimental yield test designimplemented above. Experiments were planted and trials were mechanicallycultivated and/or had labeled use rates of conventional soybeanherbicides applied, as needed, to ensure weed-free conditions. Maturityscores and yield data were collected for each entry and subject to ANOVAusing the mixed model procedure of SAS.

Event 3560.4.3.5 was selected for introgression into four differentPioneer® conventional (non-transgenic) elite lines with differentgenetic parentage. The four elite lines had relative maturities (RM) of22, 27, 30, and 38. After the initial cross, F1 seed was backcrossed tothe respective recurrent parent and BC1F1 seed was advanced to the F2generation. The four BC1F2 populations created were grown and harvestedas individual plants. BC1F3 lines were created using single plant-to-row2.4 m increase rows. Homozygous positive or homozygous negative sisterlines were selected for yield testing based upon screening of 12 remnantseedlings for each line using the chlorsulfuron herbicide seedling test.Based upon the remnant screening across the four populations, 328positive 3560.4.3.5 soybean lines, and 116 negative lines were selectedfor preliminary yield trials

For the 3560.4.3.5× elite preliminary yield trials, BC1F3 lines wereblocked by the recurrent parent and presence or absence of the3560.4.3.5 event. Positive and negative blocks were planted adjacent toeach other at different locations, based upon expected maturity of thelines within the population. The 3560.4.3.5 event positive blocks had aV3 application of 2.24 kg ai ha⁻¹ glyphosate plus 0.25% v/v NIS+2.24 kgai ha⁻¹ AMS, while negative blocks had conventional herbicides applied(as needed) to maintain weed free plots. Maturity scores and yield datawere collected for each entry at each location and subject to multipleregression and ANOVA using the GLM and mixed model procedures of SAS.

Results and Discussion

TO 3560.4.3.5 soybean were evaluated for glyphosate tolerance. The3560.4.3.5 soybean were sprayed with 2.24 kg ai ha⁻¹ glyphosate and 4.48kg ae ha⁻¹ glyphosate (Table 15). The 2.24 kg ai ha⁻¹ treatment wouldcorrespond to 2× the typical labeled field application rate ofglyphosate, while the 4.48 kg ae ha⁻¹ treatment would correspond to 4×the typical labeled field application rate of glyphosate. Theuntransformed parental line (sprayed as a control) did not surviveeither glyphosate application rate and was consistently rated a 1.

The 3560.4.3.5 event was advanced to the T1 generation in a greenhouseto confirm herbicide tolerance. The 3560.4.3.5 event had tolerance afterapplication of 1.68 kg ae ha⁻¹ glyphosate, after application of 60.0 gai ha⁻¹ thifensulfuron methyl, and after a sequential application of 70g ai ha⁻¹ thifensulfuron methyl at V3, followed by 1.68 kg ae ha⁻¹glyphosate at V5 as shown in Table 15. These data indicate event3560.4.3.5 confers tolerance to both glyphosate and thifensulfuronmethyl application at early generations after transformation.

The 3560.4.3.5 event was analyzed for Mendelian segregation across earlygenerations (Table 16).

TABLE 15 Southern copy number estimates and average visual responseratings^(†) ten days after herbicide application on T0 and T1 generationtransgenic event 3560.4.3.5 Southern Generation bands^(‡) T0 T1 Eventglyat4601 hra 1.68^(§) 3.36^(§) 1.68^(§) 60.0^(¶) 70.0 + 1.68^(#)3560.4.3.5 1 1 7.0 6.5 7.0 7.0 8.0 8.0 ^(†)Average visual responserating of tolerant plants 10 days after herbicide application in thegreenhouse (0 = dead plant to 9 = no response detected) ^(‡)Estimatednumber of copies based upon Southern analysis using HindIII and theglyat4601 gene or gm-hra gene as a probe ^(§)Glyphosate application(1.68 kg ae ha⁻¹, or 3.36 kg ae ha⁻¹) + 0.25% v/v NIS + 2.24 kg ai ha⁻¹AMS applied at the V3 growth stage ^(¶)Thifensulfuron methyl application(60.0 g ai ha⁻¹ or 70.0 g ai ha⁻¹) + 0.25% v/v NIS + 2.24 kg ai ha⁻¹ AMSapplied at the V3 growth stage. ^(#)Sequential application; initiallysprayed at the V3 growth stage with 70 g ai ha⁻¹ thifensulfuron methyl +0.25% v/v NIS + 2.24 kg ai ha⁻¹ AMS, followed by spray of 1.68 kg aeha⁻¹ glyphosate + 0.25% v/v NIS + 2.24 kg ae ha⁻¹ AMS at the V6 growthstage. The first score is 10 days after the V3 thifensulfuron methylapplication, the second score is 10 days after the V6 glyphosateapplication

TABLE 16 Segregation ratios of T1 plants and T2 progeny rows of event3560.4.3.5. Generation T1 plants^(†) T2 lines^(‡) Observed ExpectedObserved Expected Event Positive Negative Positive Negative chi² P^(§)Positive Negative Positive Negative chi² P^(§) 3560.4.3.5 59 23 61.521.1 0.605 34 9 32.3 10.3 0.609 ^(†)T1 plants were identified to bepositive or negative for the glyat transgene insert using PCRamplification ^(‡)T2 lines (derived from individual glyat positive T1plants) were screened by spraying 3.36 kg ae ha⁻¹ glyphosate plus 0.25%v/v NIS + 2.24 kg ai ha⁻¹ AMS on 12 remnant V3 plants in a greenhouse.^(§)chi² probability that deviation from expected model is due to chancealone

The T3 plants from the 3560.4.3.5 plants were sprayed in a glyphosateherbicide tolerance field trial. Table 17 shows the herbicide responseat seven DAT after application of 3.36 kg ae ha⁻¹ glyphosate, afterapplication of 6.72 kg ae ha⁻¹ glyphosate, and after application of13.44 kg ae ha⁻¹ glyphosate. The untransformed control line wassusceptible to any glyphosate application and was consistently rated asa 1.3560.4.3.5 soybean T4 plants were advanced for additional glyphosatetolerance testing. Table 17 shows the spray ratings at seven andfourteen DAT with 6.72 kg ae ha⁻¹ glyphosate.

TABLE 17 LSMeans for visual ratings^(†) of selected T3 and T4 3560.4.3.5plants, sprayed with experimental rates of glyphosate in fieldexperiments. 2004 (T3 plants)^(‡) 2005 (T4 plants)^(§) Glyphosate sprayrate (kg ae ha⁻¹) Glyphosate spray rate (kg ae ha⁻¹) Unsprayed 3.36 6.7213.44 Unsprayed 6.72 6.72 sequential^(¶) Event 7 DAT 7 DAT 7 DAT 7 DAT 7DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 3560.4.3.5 9.0 8.0 6.7 5.7 9.0 9.06.8 7.8 7.8 7.7 ^(†)Visual response ratings on a 1 to 9 scale, where 1 =completely dead plot to 9 = no response observed ^(‡)test consisted ofthree randomized replications of paired 3.7 m rows (76.2 cm row spacing)sprayed at the V3 growth stage ^(§)test consisted of two randomizedreplications of paired 1.2 m rows (76.2 cm row spacing) sprayed at theV3 growth stage ^(¶)Sequential spray; 6.72 kg ae ha⁻¹ glyphosate sprayedat V3, followed by 6.72 kg ae ha⁻¹ glyphosate sprayed on the same plotsat R1 (rating data listed below this treatment is after the R1application)

In addition to the glyphosate herbicide tolerance trials, event3560.4.3.5 was compared to a STS® cultivar in two experiments todetermine if the hra gene had better tolerance to different ALSinhibitor herbicides (Table 18). Across these two experiments, the3560.4.3.5 soybean had significantly lower crop response compared toSTS® at 7 DAT and 14 DAT after application of 8.8 g ai ha⁻¹ rimsulfuron,8.8 g ai ha⁻¹ tribenuron methyl, 4.2 g ai ha⁻¹ metsulfuron methyl, and17.5 g ai ha⁻¹ tribenuron methyl (Table 18). These chemistries arecurrently not labeled for use on soybean, and will cause high levels ofcrop response on current STS® and conventional soybean cultivars. Thetolerance data obtained indicate that 3560.4.3.5 soybean hadsignificantly higher tolerance to multiple ALS inhibitor chemistrieswhen compared directly to the STS® trait.

TABLE 18 Difference of LSMeans for crop response ratings^(†) of3560.4.3.5 soybean compared to STS ® across different ALS inhibitorherbicide treatments in experiments. Rate 7 DAT 14 DAT Treatment^(‡) (gai ha⁻¹) Location^(§) Reps 3560.4.3.5 STS ® Difference Pr > |t|3560.4.3.5 STS ® Rimsulfuron 8.8 A 2 10.0 50.0 −40.0 <.0001 5.0 55.0Tribenuron methyl 8.8 A 2 5.0 35.0 −30.0 <.0001 5.0 42.5 Chlorimuronethyl + 30.0 + 9.67 A 2 0.0 5.0 −5.0 0.200 0.0 5.0 thifensulfuron methylUnsprayed control 0 A 2 0.0 0.0 0.0 1.000 0.0 0.0 Metsulfuron methyl 4.2B 6 58.3 79.2 −20.8 <.0001 64.2 90.8 Thifensulfuron methyl 70 B 6 23.327.5 −4.2.0 0.549 6.7 8.3 Tribenuron methyl 17.5 B 6 11.7 36.7 −25.0<.0001 15.0 48.3 Unsprayed control 0 B 6 0.0 0.0 0.0 1.000 1.7 0.0 14DAT 28 DAT^(¶) Treatment^(‡) Difference Pr > |t| 3560.4.3.5 STS ®Difference Pr > |t| Rimsulfuron −50.0 <.0001 Tribenuron methyl −37.5<.0001 Chlorimuron ethyl + −5.0 0.171 thifensulfuron methyl Unsprayedcontrol −45.0 <.0001 Metsulfuron methyl −26.7 <.0001 29.2 95.0 −65.8<.0001 Thifensulfuron methyl −1.7 0.797 8.3 6.7 1.7 0.803 Tribenuronmethyl −33.3 <.0001 5.0 21.7 −16.7 0.015 Unsprayed control 1.7 0.747 1.70.0 1.7 0.804 ^(†)Visual response ratings from 0% to 100%, where 0% = noresponse observed to 100% = plot completely dead. ^(‡)0.25% v/v NIS +2.24 kg ai ha⁻¹ AMS was added to all spray treatments ^(§)A locationconsisted of two replications of paired 3.7 m rows (76 cm row spacing);B location consisted of three replications of paired 3.7 m rows (76.2 cmrow spacing), grown at two different locations. ^(¶)Due to minimalresponse of 35604.3.5 at 14 DAT, the 28 DAT ratings were not recordedfor the A experimentEvent 3560.4.3.5 was evaluated for herbicide tolerance and yieldperformance in more extensive testing. For the first set of experiments,event 3560.4.3.5 was sprayed at a vegetative stage and a reproductivestage with different tank mixes of glyphosate and other pesticides.Across the treatments evaluated, there was minimal crop response at 7DAT and 14 DAT (10% or less) for the glyphosate and all glyphosate pluspesticide mixtures applied at the V2 growth stage, except thosecontaining bentazon or fomesafen (Table 19). The treatments withbentazon or fomesafen caused up to 20% initial phytotoxicity at 7 DAT,which diminished to less than 12% by 14 DAT. These results wereexpected, as the leaf bronzing observed was equivalent to phytotoxicitythat is typically observed on commodity soybeans up to 14 days afterapplication with either bentazon of fomesafen chemistry. It should alsobe noted that herbicide response ratings are very subjective, and ingeneral, a 10% response cannot be easily distinguished unless there isan unsprayed control right next to the plot being evaluated. By 28 DAT,there was essentially no crop response to any of the treatments appliedat the V2 growth stage (Table 19). For the mixtures applied at R2, allof the treatments had 10% or less response at 7 DAT, 14 DAT, and 28 DATexcept for the treatment containing bentazon (Table 19). For thistreatment an average crop response of 11.4% was recorded at 7 DAT, whichdiminished to less than 5% at 14 DAT and 0% at 28 DAT (Table 19). Thecrop response observed was the typical leaf bronzing that is commonlyobserved up to 14 DAT after application of bentazon on commoditysoybeans. In general, 3560.4.3.5 soybean had excellent tolerance todifferent mixtures of glyphosate with and without other pesticidesacross two different growth stages evaluated in different environments.

TABLE 19 LSMeans for crop response ratings^(†) of 3560.4.3.5 soybeantreated at two different growth stages with different tank mixedpesticide formulations. Glyphosate Other pesticides Application 7 DAT 14DAT 28 DAT Treatment^(‡) rate (kg ae ha−1) rate (g ai ha⁻¹) Stage RepsAverage Average Average Glyphosate 3.36 V2 9 3.9 3.3 0.6 Glyphosate +chlorpyrifos 3.36 560.4 V2 9 5.6 3.3 0.0 Glyphosate + bentazon 3.361120.9 V2 9 16.6 6.3 0.6 Glyphosate + imazethapyr 3.36 141.3 V2 9 5.02.2 0.6 Glyphosate + fomesafen 3.36 263.4 V2 9 20.3 12.0 0.6Glyphosate + tribenuron-methyl + thifensulfuron-methyl 3.36 17.5 + 17.5V2 9 5.0 8.3 0.0 Glyphosate + tribenuron-methyl + 3.36 17.5 + 17.5 +560.4 V2 9 7.6 10.8 0.0 thifensulfuron-methyl + chlorpyrifosGlyphosate + tribenuron-methyl + 3.36 17.5 + 17.5 + 1120.9 V2 9 17.9 5.00.0 thifensulfuron-methyl + bentazon Glyphosate + tribenuron-methyl +3.36 17.5 + 17.5 + 141.3 V2 9 5.1 7.8 0.0 thifensulfuron-methyl +imazethapyr Glyphosate + tribenuron-methyl + 3.36 17.5 + 17.5 + 263.4 V29 19.8 11.4 1.1 thifensulfuron-methyl + fomesafen Glyphosate 3.36 R2 91.7 0.6 0.0 Glyphosate + chlorpyrifos 3.36 560.4 R2 9 1.7 0.6 0.0Glyphosate + bentazon 3.36 1120.9 R2 9 11.4 2.2 0.0 Glyphosate +imazethapyr 3.36 141.3 R2 9 0.6 0.0 0.0 Glyphosate + fomesafen 3.36263.4 R2 9 9.6 1.7 0.0 Glyphosate + tribenuron-methyl +thifensulfuron-methyl 3.36 17.5 + 17.5 R2 9 1.1 0.0 0.0 Glyphosate +tribenuron-methyl + 3.36 17.5 + 17.5 + 560.4 R2 9 2.8 0.0 0.0thifensulfuron-methyl + chlorpyrifos Glyphosate + tribenuron-methyl +3.36 17.5 + 17.5 + 1120.9 R2 9 7.0 1.7 0.0 thifensulfuron-methyl +bentazon Glyphosate + tribenuron-methyl + 3.36 17.5 + 17.5 + 141.3 R2 91.7 0.0 0.0 thifensulfuron-methyl + imazethapyr Glyphosate +tribenuron-methyl + 3.36 17.5 + 17.5 + 263.4 R2 9 5.3 2.8 0.0thifensulfuron-methyl + fomesafen Unsprayed control 3.36 R2 9 0.0 0.00.0 LSD (a = 0.05) 3.3 3.3 2.0 ^(†)Visual response ratings from 0% to100%, where 0% = no response observed to 100% = plot completely dead.^(‡)All treatments had 0.25% v/v NIS + 2.24 kg ai ha⁻¹ AMS added.

The second set of experiments conducted with event 3560.4.3.5 weredesigned to measure yield potential after application of differentherbicides at field use rates that would be commonly utilized forsoybean production. Across the glyphosate, ALS inhibitor, and glyphosateplus ALS inhibitor treatments there was minimal (10% or less) toessentially no response at 7, 14, and 28 DAT (Table 20). When yield datawas collected, there were no significant yield differences detected forany of the herbicide treatments applied at different the growth stages(Table 20). In addition, the replications that received herbicidetreatments were not statistically different from the unsprayed controlreplications for yield (Table 20). Based upon multiple year herbicidetolerance trials, it can be concluded that event 3560.4.3.5 hadexcellent tolerance to field use rates of glyphosate, ALS inhibitor, andglyphosate plus ALS inhibitor herbicides. In addition, yield potentialwas not affected when event 3560.4.3.5 was sprayed with glyphosate, ALSinhibitor, and glyphosate plus ALS inhibitor chemistries at differentvegetative and reproductive growth stages.

TABLE 20 LSMeans for crop response ratings^(†) and yield (kg ha⁻¹) for3560.4.3.5 soybean treated at four different growth stages withdifferent herbicide combinations. Glyphosate ALS inhibitor Applicationrate (kg ae ha⁻¹) rate (g ai ha⁻¹) Stage Reps Treatment^(‡) 7 DAT 14 DAT28 DAT Yield Glyphosate 1.68 VC 18 1.6 0.8 0.6 2745.7 Glyphosate 1.68 V218 5.3 2.8 0.6 2851.7 Glyphosate 1.68 R2 18 2.0 0.6 0.0 2702.9Glyphosate 1.68 R5 18 0.3 0.3 0.0 2718.8 Chlorimuron ethyl +thifensulfuron methyl 5.8 + 4.4 VC 18 0.7 0.6 0.6 2756.4 Chlorimuronethyl + thifensulfuron methyl 5.8 + 4.4 V2 18 0.6 0.0 0.3 2864.0Chlorimuron ethyl + thifensulfuron methyl 5.8 + 4.4 R2 18 0.0 0.0 0.32899.2 Chlorimuron ethyl + thifensulfuron methyl 5.8 + 4.4 R5 18 0.0 0.00.6 2861.1 Glyphosate + chlorimuron ethyl + thifensulfuron methyl 1.685.8 + 4.4 VC 18 2.6 1.7 0.0 2647.3 Glyphosate + chlorimuron ethyl +thifensulfuron methyl 1.68 5.8 + 4.4 V2 18 6.9 3.8 2.2 2829.7Glyphosate + chlorimuron ethyl + thifensulfuron methyl 1.68 5.8 + 4.4 R218 0.8 0.0 0.3 2841.6 Glyphosate + chlorimuron ethyl + thifensulfuronmethyl 1.68 5.8 + 4.4 R5 18 1.1 0.0 0.0 2768.3 Hand weeded controlControl 18 0.0 0.0 0.0 2813.1 Unsprayed control Control 18 0.0 0.0 0.02742.4 LSD (a = 0.05) 1.3 1.1 0.8 155.6 ^(†)Visual response ratings from0% to 100%, where 0% = no response observed to 100% = plot completelydead ^(‡)Two different field locations were utilized; one field hadsevere drought stress during the spring and early summer, the other waswell irrigated. ^(‡)All treatments had 0.25% v/v NIS + 2.24 kg ai ha⁻¹AMS added

Isoline yield test data were collected in five additional environmentsfor event 3560.4.3.5 soybean. When isoline yield data were subject tomixed model ANOVA, the year, and location (nested within year) weresignificantly different (Table 21). Positive 3560.4.3.5 soybean andnegative NILs, and the interactions with the 3560.4.3.5 event were notsignificantly different for yield (Table 21). These data indicate thatpresence of event 3560.4.3.5 does not impact final yield potential whenNILs were tested across multiple years and environments.

TABLE 21 Mixed model ANOVA for yield (kg ha⁻¹) of positive and negativeNILs of event 3560.4.3.5, tested in fourteen environments. Type 3 Testsof Fixed Effects Effect Num DF Den DF F Value Pr > F Year 2 80 26.92<.0001 Location (Year) 11 80 7.82 <.0001 glyat + hra 1 80 0.04 0.850Year * glyat + hra 2 80 0.63 0.533 Location (Year) * glyat + hra 11 801.75 0.077

Event 3560.4.3.5 was backcrossed into four different Pioneer®conventional elite lines to confirm Mendelian segregation, and tomeasure yield impact of the event in different genetic backgrounds. Foreach of the four different BC1F2 populations tested, the seedlingssegregated in a 3:1 (tolerant:susceptible) ratio, when analyzed usingchlorsulfuron screening (Table 22). When event 3560.4.3.5 was forwardcrossed into 31 different genetic backgrounds, all F2 populationsexamined segregated in a 3:1 (tolerant:susceptible) ratio when sprayedin the field with glyphosate (Table 22). These data suggest that indifferent genetic backgrounds, event 3560.4.3.5 will confer tolerance toglyphosate and ALS inhibitor herbicides, and will segregate as a singledominant gene.

TABLE 22 F2 segregation ratios of event 3560.4.3.5 backcrossed into fourdifferent elite genetic backgrounds, and forward crossed into 31different elite genetic backgrounds. plants observed plants expectedPopulation Generation^(†) Resistant Susceptible Resistant SusceptibleP^(‡) 3560.4.3.5x Elite7 BC1F2 700 222 691.5 230.5 0.518 3560.4.3.5xElite8 BC1F2 761 273 775.5 258.5 0.298 3560.4.3.5x Elite9 BC1F2 160 54160.5 53.5 0.937 3560.4.3.5x Elite10 BC1F2 205 79 213.0 71.0 0.2733560.4.3.5x PHI1 F2 53 23 57.0 19.0 0.289 3560.4.3.5x PHI2 F2 76 20 72.024.0 0.346 3560.4.3.5x PHI3 F2 66 24 67.5 22.5 0.715 3560.4.3.5x PHI4 F275 23 73.5 24.5 0.726 3560.4.3.5x PHI5 F2 99 23 91.5 30.5 0.1173560.4.3.5x PHI6 F2 96 24 90.0 30.0 0.206 3560.4.3.5x PHI11 F2 72 1867.5 22.5 0.273 3560.4.3.5x PHI12 F2 115 36 113.3 37.8 0.742 3560.4.3.5xPHI13 F2 91 22 84.8 28.3 0.175 3560.4.3.5x PHI14 F2 97 26 92.3 30.80.323 3560.4.3.5x PHI15 F2 88 28 87.0 29.0 0.830 3560.4.3.5x PHI16 F2 6624 67.5 22.5 0.715 3560.4.3.5x PHI17 F2 75 34 81.8 27.3 0.1353560.4.3.5x PHI18 F2 108 25 99.8 33.3 0.099 3560.4.3.5x PHI19 F2 78 2678.0 26.0 1.000 3560.4.3.5x PHI20 F2 95 29 93.0 31.0 0.678 3560.4.3.5xPHI21 F2 109 37 109.5 36.5 0.924 3560.4.3.5x PHI22 F2 75 26 75.8 25.30.863 3560.4.3.5x PHI23 F2 85 19 78.0 26.0 0.113 3560.4.3.5x PHI24 F2 8118 74.3 24.8 0.117 3560.4.3.5x PHI25 F2 76 23 74.3 24.8 0.6853560.4.3.5x PHI26 F2 97 36 99.8 33.3 0.582 3560.4.3.5x PHI27 F2 98 2894.5 31.5 0.471 3560.4.3.5x PHI28 F2 89 22 83.3 27.8 0.208 3560.4.3.5xPHI29 F2 71 24 71.3 23.8 0.953 3560.4.3.5x PHI30 F2 54 15 51.8 17.30.532 3560.4.3.5x PHI31 F2 73 20 69.8 23.3 0.436 3560.4.3.5x PHI32 F2 5312 48.8 16.3 0.223 3560.4.3.5x PHI33 F2 96 26 91.5 30.5 0.3473560.4.3.5x PHI34 F2 76 20 72.0 24.0 0.346 3560.4.3.5x PHI35 F2 69 1865.3 21.8 0.353 ^(†)BC1F2 populations were screened using chlorsulfuronsolution on emerging seedlings. F2 populations were grown in the fieldand sprayed with 2.24 kg ai ha⁻¹ glyphosate + 0.25% v/v NIS + 2.24 kg aiha⁻¹ AMS at the V4 growth stage. Resistant and susceptible plants werecounted approximately 7 days after treatment for all populationsexamined. ^(‡)chi² probability that deviation from expected model is dueto chance alone

To test the yield impact of event 3560.4.3.5 in different geneticbackgrounds, BC1F3 lines were developed that were either homozygouspositive or homozygous negative for the 3560.4.3.5 event (Table 23).Since these lines are not true NILs, there may be some confounding errorassociated with the preliminary yield tests evaluated. For example,later maturing soybean lines typically have higher yield compared toearlier maturing lines within the same population. Therefore, maturityfor each BC1F3 line was calculated as the number of days from plantingto the estimated R8 growth stage (date when 95% of the pods had reachedtheir mature pod color). Maturity estimates for each line were developedbased upon direct comparison at each location to several non-transgenicPioneer® experimental lines of known maturity. For the preliminary yieldtrail data analysis, maturity was analyzed as a covariate by nestingwithin the 3560.4.3.5 event to allow for a yield comparison between3560.4.3.5 event positive and 3560.4.3.5 event negative lines at thesame estimated maturity. It should be noted that the BC1F3 glyat+hrapositive versus glyat+hra negative comparisons reported may also beconfounded by the different herbicide programs utilized. However,blocking conventional cultivar trials from glyphosate tolerant trialshas been a common practice in soybean cultivar development programs. Inaddition, for the 3560.4.3.5× elite data analysis presented, it wasassumed that segregation of background maturity alleles, diseaseresistance alleles, and all other background genetic effects would occurat the same frequency within a population of 3560.4.3.5 event positivelines and within a population of 3560.4.3.5 event negative lines derivedfrom the same initial BC1F1 plant.

Yield LSMeans of 3560.4.3.5 event positive and 3560.4.3.5 event negativelines were examined for each of the four BC1F3 populations tested acrossdifferent locations (Table 23). For the BC1F3 population of3560.4.3.5×RM22 Elite, the 3560.4.3.5 event positive lines withinmaturity group 113 had significantly higher yield compared to the3560.4.3.5 event negative lines within maturity group 113 (Table 23).This difference was most likely due to environmental effect, as for allthe other maturity groupings, 3560.4.3.5 event positive and 3560.4.3.5event negative lines were not statistically different for yield (Table23). In addition, at a specific location, and when yield data from alllocations tested were pooled, 3560.4.3.5 event positive lines were notsignificantly different for yield compared to 3560.4.3.5 event negativelines within the 3560.4.3.5×RM22 Elite population (Table 23).

For the population of 3560.4.3.5×RM27 Elite BC1F3 lines, the 3560.4.3.5event positive lines tested had a significant yield advantage comparedto the 3560.4.3.5 event negative lines (Table 23). This observation ismost likely due to an environmental effect, as when all locations werepooled, there were no significant differences detected for yield between3560.4.3.5 event positive and 3560.4.3.5 event negative lines (Table23). In addition, there were no significant differences detected foryield when 3560.4.3.5 event positive lines were compared to 3560.4.3.5event negative lines at each specific maturity grouping (Table 23).

At two locations, there were significant yield differences observedwithin the population of 3560.4.3.5×RM30 Elite BC1F3 lines. 3560.4.3.5event positive lines had significantly higher yield compared to3560.4.3.5 event negative lines, while at a different location, theopposite effect was noted (Table 23). When all locations are pooled,there was not a significant difference detected for yield between3560.4.3.5 event positive and 3560.4.3.5 event negative lines (Table23). These results suggest environmental influence was most likelycausing the effect, as at each specific maturity grouping, there were nosignificant differences detected for yield when 3560.4.3.5 eventpositive lines were directly compared to 3560.4.3.5 event negativesister lines (Table 23).

Two significant yield differences were also observed within thepopulation of 3560.4.3.5×RM38 Elite BC1F3 lines at specific locations.In one of the replications, 3560.4.3.5 event positive lines hadsignificantly higher yield compared to glyat+hra negative lines, whilein another location, 3560.4.3.5 event negative lines had a yieldadvantage (Table 23). There was not a significant difference detectedfor yield when all locations were pooled for this population, whichsuggested environmental influence within a single location is causingthe difference. At each of the maturity groupings, there was only asignificant yield effect detected at maturity group 125, and in thatcase the 3560.4.3.5 event positive lines had significantly higher yieldcompared to 3560.4.3.5 event negative lines (Table 23). No distinctyield trends were evident across the four populations tested, indicatingpresence of the 3560.4.3.5 event does not impact yield potential whenintegrated into different genetic backgrounds.

TABLE 23 LSMeans for yield (kg ha⁻¹) at a location and a specificmaturity for homozygous 3560.4.3.5 event positive and 3560.4.3.5 eventnegative BC1F3 lines within four different populations of event3560.4.3.5x elite backgrounds. 3560.4.3.5 event Population Location^(†)Maturity^(‡) Positive Negative Difference^(§) Pr > |t| 3560.4.3.5x RM22ELITE A All 2471.74 2439.77 31.97 0.675 3560.4.3.5x RM22 ELITE B All3491.77 3393.32 98.45 0.193 3560.4.3.5x RM22 ELITE C All 2683.63 2747.03−63.40 0.402 3560.4.3.5x RM22 ELITE D All 3180.61 3144.42 36.19 0.6323560.4.3.5x RM22 ELITE E All 2996.18 2912.63 83.55 0.271 3560.4.3.5xRM22 ELITE All All 2964.78 2927.43 37.35 0.341 3560.4.3.5x RM22 ELITEAll 111 2801.79 2836.18 −34.39 0.807 3560.4.3.5x RM22 ELITE All 1122927.15 2704.48 222.68 0.072 3560.4.3.5x RM22 ELITE All 113 2881.952686.65 195.30* 0.041 3560.4.3.5x RM22 ELITE All 114 2625.92 2811.09−185.17 0.285 3560.4.3.5x RM22 ELITE All 115 2899.19 2839.28 59.91 0.6333560.4.3.5x RM22 ELITE All 116 2848.24 2859.77 −11.53 0.920 3560.4.3.5xRM22 ELITE All 125 3183.30 2969.74 213.56 0.231 3560.4.3.5x RM22 ELITEAll 126 2976.31 3045.30 −69.00 0.770 3560.4.3.5x RM22 ELITE All 1272853.20 3029.97 −176.77 0.327 3560.4.3.5x RM22 ELITE All 128 3031.553030.07 1.48 0.986 3560.4.3.5x RM22 ELITE All 129 3088.60 2994.95 93.650.350 3560.4.3.5x RM22 ELITE All 130 3198.18 3166.12 32.06 0.8033560.4.3.5x RM22 ELITE All 131 3117.01 2981.93 135.08 0.435 3560.4.3.5xRM22 ELITE All 132 3074.62 3028.53 46.09 0.705 3560.4.3.5x RM27 ELITE BAll 3331.34 3406.61 −75.26 0.640 3560.4.3.5x RM27 ELITE C All 3218.792886.77 332.02* 0.014 3560.4.3.5x RM27 ELITE D All 3475.08 3417.17 57.910.656 3560.4.3.5x RM27 ELITE E All 3028.48 3149.72 −121.24 0.5303560.4.3.5x RM27 ELITE F All 3099.48 3021.84 77.64 0.600 3560.4.3.5xRM27 ELITE All All 3230.63 3176.42 54.21 0.487 3560.4.3.5x RM27 ELITEAll 122 3311.82 3145.60 166.22 0.497 3560.4.3.5x RM27 ELITE All 1233226.68 3164.15 62.53 0.668 3560.4.3.5x RM27 ELITE All 125 3148.803238.51 −89.71 0.660 3560.4.3.5x RM27 ELITE All 126 3086.18 3213.06−126.88 0.449 3560.4.3.5x RM27 ELITE All 127 3271.69 3168.42 103.280.670 3560.4.3.5x RM27 ELITE All 128 3237.08 3106.81 130.27 0.4363560.4.3.5x RM27 ELITE All 130 3127.15 2987.23 139.92 0.594 3560.4.3.5xRM27 ELITE All 133 3435.66 3387.58 48.08 0.843 3560.4.3.5x RM30 ELITE CAll 3298.88 2983.11 315.77* <.0001 3560.4.3.5x RM30 ELITE D All 3544.233693.09 −148.86* 0.029 3560.4.3.5x RM30 ELITE G All 3472.84 3481.88−9.05 0.888 3560.4.3.5 x RM30 ELITE E All 3177.69 3229.61 −51.92 0.4183560.4.3.5x RM30 ELITE F All 3002.89 2975.75 27.13 0.673 3560.4.3.5xRM30 ELITE All All 3299.30 3272.69 26.62 0.512 3560.4.3.5x RM30 ELITEAll 123 3356.46 3010.13 346.33 0.063 3560.4.3.5x RM30 ELITE All 1253276.10 3172.47 103.63 0.194 3560.4.3.5x RM30 ELITE All 126 3266.423168.61 97.81 0.228 3560.4.3.5x RM30 ELITE All 127 3297.75 3299.10 −1.350.984 3560.4.3.5x RM30 ELITE All 128 3285.84 3282.21 3.63 0.9343560.4.3.5x RM30 ELITE All 129 3278.27 3407.86 −129.59 0.051 3560.4.3.5xRM30 ELITE All 130 3334.29 3568.44 −234.15 0.118 3560.4.3.5x RM38 ELITEC All 2734.84 2659.09 75.75 0.256 3560.4.3.5x RM38 ELITE G All 3228.833305.91 −77.08 0.248 3560.4.3.5x RM38 ELITE E All 3049.84 3190.55−140.71* 0.040 3560.4.3.5x RM38 ELITE F1 All 2678.11 2611.45 66.65 0.3443560.4.3.5x RM38 ELITE F2 All 2687.71 2507.70 180.02* 0.008 3560.4.3.5xRM38 ELITE All All 2875.87 2854.94 20.93 0.608 3560.4.3.5x RM38 ELITEAll 122 2700.19 2803.14 −102.96 0.547 3560.4.3.5x RM38 ELITE All 1242743.61 2824.11 −80.50 0.564 3560.4.3.5x RM38 ELITE All 125 3206.302880.53 325.77* 0.001 3560.4.3.5x RM38 ELITE All 126 2788.26 2864.36−76.10 0.550 3560.4.3.5x RM38 ELITE All 127 2849.14 2929.84 −80.71 0.5143560.4.3.5x RM38 ELITE All 128 2842.31 2989.39 −147.07 0.306 3560.4.3.5xRM38 ELITE All 129 2928.44 2884.00 44.44 0.490 3560.4.3.5x RM38 ELITEAll 130 2918.29 2864.10 54.19 0.325 3560.4.3.5x RM38 ELITE All 1312906.27 2655.01 251.26 0.057 ^(‡)Maturity is calculated as the averagenumber of days between planting and R8 growth stage for the plot.^(§)Estimated yield LSMean difference between 3560.4.3.5 event positiveand 3560.4.3.5 event negative lines. *Indicates estimated yielddifference is significant at P = 0.05.

TABLE 24 Description of Genetic Elements in Fragment PHP20163A Locationon fragment PHP20163A Size (base pair Genetic (base position) Elementpairs) Description  1 to 16 polylinker 16 Region for cloning geneticelements region  17 to 502 SCP1 promoter 486 Constitutive syntheticpromoter comprising a portion of the CaMV 35S promoter (Odell et al.(1985) Nature 313: 801-812 and the Rsyn7-Syn II Core consensus promoter(U.S. Pat. No. 6,0720,050 and 6,555,673). 503 to 504 polylinker 2 Regionfor cloning genetic elements region 505 to 571 TMV omega 5′- 67 Anenhancer element derived from the Tobacco UTR Mosaic Virus omega 5′untranslated leader (Gallie and Walbot (1992) NAR 20: 4631-4638. 572 to596 polylinker 25 Region for cloning genetic elements region  597 to1037 glyat4601 gene 441 Synthetic glyphosate N-acetyltransferase (glyat)gene (Castle et al. (2004) Science 304: 1151-1154). 1038 to 1053polylinker 16 Region for cloning genetic elements region 1054 to 1369pinII terminator 316 Terminator region from Solanum tuberosum proteinaseinhibitor II (pinII) gene (Keil et al. (1986) NAR 14: 5641-5650; An etal. (1989) Plant Cell 1: 115-122. 1370 to 1385 polylinker 16 Region forcloning genetic elements region 1386 to 2030 SAMS promoter 645 Promoterof the S-adenosyl-L-methionine synthetase (SAMS) gene from soybean(Falco and Li (2003) U.S. publication 2003/0,226,166. 2031 to 2089 SAMS5′-UTR 59 5′ untranslated region of the SAMS gene from soybean (Falcoand Li, 2003 U.S. publication 2003/0,226,166). 2090 to 2680 SAMS intron591 Intron within the 5′-untranslated region of the SAMS gene fromsoybean (Falco and Li, 2003 U.S. publication 2003/0,226,166). 2681 to2696 SAMS 5′-UTR 16 5′ untranslated region (UTR) of the SAMS gene fromsoybean (Falco and Li, 2003 U.S. publication 2003/0,226,166). 2697 to4667 gm-hra gene 1971 Modified version of the acetolactate synthase genefrom soybean with 15 additional nucleotides on the 5′ end (2697 to 2711)derived from the SAMS gene and two nucleotide changes within the codingsequence. 4668 to 5319 als terminator 652 Native terminator from thesoybean acetolactate synthase gene. 5319 to 5362 polylinker 43 Regionfor cloning genetic elements region

Example 4 Improved Yield of Soybean Event 3560.4.3-5 in Ten PopulationsAdapted for the Southern Growing Region of the United States

Soybean with the GLYAT gene from event 3560.4.3.5 and an EPSPS genecorresponding to the EPSPS described in S. R. Pagette et al (1995)Development, Identification, and Characterization of aGlyphosate-Tolerance Soybean Line. Crop Sci. 35:1451-1461 (hereinincorporated by reference) were generated. The EPSPS event of theglyphosate-tolerant soybean line 40-3-2 and the GLYAT event of theglyphosate-tolerant soybean line 3560.4.3.5 were brought together viaconventional breeding to generate ten unique populations. The lines foreach population were identified as containing the GLYAT event3560.4.3.5, the EPSPS event 40-3-2, or containing both the GLYAT andEPSPS events. Lines were grown in the summer season as a plant row yieldtrials (PRYT) near West Memphis, Ark. PRYT rows were 1.2 meters inlength, with 76 cm between row spacing. Plots were sprayed 24 daysfollowing planting with 840 g ae/ha glyphosate and sprayed 44 days afterplanting with 1680 g ae/ha glyphosate. Maturity and yield data werecollected for each line and analyzed using the PROC Mixed function ofSAS (SAS Institute, Cary N.Y.). Yields were adjusted for maturity forvalid comparisons. When pooling the three classes (GLYAT, EPSPS,GLYAT+EPSPS) over the ten populations, the GLYAT+EPSPS lines weresignificantly higher yielding (48.1 bu/acre) compared to the EPSPS lines(40.6 bu/acre) and the GLYAT lines (44.7 bu/ac).

Table 25 shows the differences between LSMean estimates (bu/ac) foryield of ten different populations of related lines classified forglyphosate tolerance transgenes (GLYAT, EPSPS, GLYAT+EPSPS). FIG. 11provides LSMean comparisons for yield (bu/ac) of ten differentpopulations of lines classified for glyphosate tolerance transgenes(GLYAT, EPSPS, GLYAT+EPSPS). Lines were adapted to the Southern UnitedStates growing region.

TABLE 25 Population Herbicide1 n1 LSMean1 Herbicide2 n2 LSMean2Difference Probt All GLYAT 80 44.7 GLYAT + EPSPS 575 48.1 −3.5 0.032 AllGLYAT 80 44.7 EPSPS 129 40.6 4.0 0.040 All GLYAT + EPSPS 575 48.1 EPSPS129 40.6 7.5 0.000 Population1 GLYAT 6 36.3 GLYAT + EPSPS 64 43.8 −7.5NS Population1 GLYAT 6 36.3 EPSPS 18 43.9 −7.6 NS Population1 GLYAT +EPSPS 64 43.8 EPSPS 18 43.9 −0.1 NS Population2 GLYAT 6 41.8 GLYAT +EPSPS 29 42.3 −0.5 NS Population2 GLYAT 6 41.8 EPSPS 8 31.6 10.2 NSPopulation2 GLYAT + EPSPS 29 42.3 EPSPS 8 31.6 10.7 0.035 Population3GLYAT 9 47.6 GLYAT + EPSPS 88 51.7 −4.1 NS Population3 GLYAT 9 47.6EPSPS 21 47.1 0.4 NS Population3 GLYAT + EPSPS 88 51.7 EPSPS 21 47.1 4.6NS Population4 GLYAT 6 33.8 GLYAT + EPSPS 43 39.1 −5.2 NS Population4GLYAT 6 33.8 EPSPS 5 25.2 8.6 NS Population4 GLYAT + EPSPS 43 39.1 EPSPS5 25.2 13.9 0.021 Population5 GLYAT 4 49.5 GLYAT + EPSPS 37 43.9 5.6 NSPopulation5 GLYAT 4 49.5 EPSPS 12 35.1 14.4 0.049 Population5 GLYAT +EPSPS 37 43.9 EPSPS 12 35.1 8.8 0.036 Population6 GLYAT 9 52.7 GLYAT +EPSPS 32 51.6 1.0 NS Population6 GLYAT 9 52.7 EPSPS 10 49.9 2.8 NSPopulation6 GLYAT + EPSPS 32 51.6 EPSPS 10 49.9 1.7 NS Population7 GLYAT12 41.8 GLYAT + EPSPS 87 43.2 −1.3 NS Population7 GLYAT 12 41.8 EPSPS 1038.3 3.5 NS Population7 GLYAT + EPSPS 87 43.2 EPSPS 10 38.3 4.9 NSPopulation8 GLYAT 13 51.7 GLYAT + EPSPS 61 53.6 −1.9 NS Population8GLYAT 13 51.7 EPSPS 24 45.5 6.2 NS Population8 GLYAT + EPSPS 61 53.6EPSPS 24 45.5 8.0 0.008 Population9 GLYAT 10 50.7 GLYAT + EPSPS 70 61.1−10.4 0.015 Population9 GLYAT 10 50.7 EPSPS 10 43.7 7.0 NS Population9GLYAT + EPSPS 70 61.1 EPSPS 10 43.7 17.4 0.000 Population10 GLYAT 5 40.6GLYAT + EPSPS 64 51.0 −10.4 NS Population10 GLYAT 5 40.6 EPSPS 11 46.0−5.4 NS Population10 GLYAT + EPSPS 64 51.0 EPSPS 11 46.0 5.0 NS

Example 5 Improved Yield of Soybean Event 3560.4.3.5 in Two PopulationsAdapted for the Mid-Maturity Growing Region of the United States

Soybean with the 3560.4.3.5 event and an EPSPS gene corresponding to theEPSPS described in S. R. Pagette et al (1995) Development,Identification, and Characterization of a Glyphosate-Tolerance SoybeanLine. Crop Sci. 35:1451-1461 (herein incorporated by reference) weregenerated. The EPSPS event of the glyphosate-tolerant soybean line40-3-2 and the GLYAT event of the glyphosate-tolerant soybean line3560.4.3.5 were brought together via conventional breeding to generatetwo unique populations. The lines for each population were identified ascontaining the GLYAT event 3560.4.3.5, the EPSPS event 40-3-2, orcontaining both the GLYAT and EPSPS events. Lines were grown in thesummer season as a plant row yield trials (PRYT) near Napoleon, Ohio.PRYT rows were 1.2 meters in length, with 76 cm between row spacing.Plots were sprayed 31 days after planting with 3360 g ae/ha glyphosate.Maturity and yield data were collected for each line and analyzed usingthe PROC Mixed function of SAS (SAS Institute, Cary N.Y.). Yields wereadjusted for maturity for valid comparisons. When pooling the threeacross the two populations, the GLYAT+EPSPS lines were significantlyhigher yielding (45.6 bulacre) compared to the EPSPS lines (41.4bu/acre) and not significantly different compared to the GLYAT lines(45.8 bu/acre).

Table 26 shows the differences between LSMean estimates for yield of twodifferent populations of lines classified for glyphosate tolerancetransgenes (GLYAT, EPSPS, GLYAT+EPSPS). FIG. 12 provides LSMeancomparisons for yield of two different populations of related linesclassified for glyphosate tolerance transgenes (GLYAT, EPSPS,GLYAT+EPSPS). Lines are adapted to the Midwestern United States growingregion.

TABLE 26 Yield Yield Comparison LSMean1 Comparison LSMean2 DifferencePopulation Class 1 N1 Bu/acre Class 2 N2 Bu/acre Bu/acre Probt All GLYAT619 45.8 GLYAT + 95 45.6 0.2 NS EPSPS All GLYAT 619 45.8 EPSPS 21 41.44.4 0.016 All GLYAT + 95 45.6 EPSPS 21 41.4 4.1 0.043 EPSPS Population1GLYAT 219 49.2 GLYAT + 72 47.9 1.3 NS EPSPS Population1 GLYAT 219 49.2EPSPS 10 47.6 1.6 NS Population1 GLYAT + 72 47.9 EPSPS 10 47.6 0.3 NSEPSPS Population2 GLYAT 400 42.4 GLYAT + 23 43.3 −0.9 NS EPSPSPopulation2 GLYAT 400 42.4 EPSPS 11 35.3 7.1 0.004 Population2 GLYAT +23 43.3 EPSPS 11 35.3 8.0 0.008 EPSPS

TABLE 27 Summary Table of SEQ ID NOS SEQ ID NO Description 1 Leftsequence junction SC36 2 Right sequence junction D32ALS 3 completeinserted transgene 4 Left genomic border 5 Right genomic border 6complete flanking and complete transgene insert 7 Forward primer 8Reverse primer 9 Taqman MGB probe 10 Left flanking genomic/left bordertransgene (10 nt/10 nt) 11 Right flanking genomic/right border transgene(10 nt/10 nt) 12 Left flanking genomic/left bordertransgene (20 nt/20nt) 13 Right flanking genomic/right border transgene (20 nt/20 nt) 14Left flanking genomic/5′ transgene 15 Right flanking genomic/3′transgene 16 Primer 1297 17 Primer 1298 18 Primer 1439 19 Primer 1514 20Primer 1558 21 Primer 1660 22 Primer 1666 23 Primer 1679 24 Primer 122725 Primer 1297 26 Primer 1440 27 left flanking genomic/left bordertransgene (30 nt/30 nt) 28 Right flanking genomic/right border transgene(30 nt/30 nt) 29 SCP1 promoter probe 30 glyat4601 probe 31 pinIIterminator probe 32 SAMS probe (5′ end) 33 SAMS probe (3′ end) 34 gm-hraprobe (5′ end) 35 gm-hra probe (3′ end) 36 als probe 37 Primer DP-356-f138 Primer DP-356-r1 39 Primer DP-356-p 40 Primer 1610 41 Left flankinggenomic/transgene 42 Right flanking genomic/transgene 43 181 nucleotidesof the 5′ end of transgene insert 44 Primer 104312 45 Primer 104314 46Probe 125323 47 Primer 109893 (endogenous control) 48 Primer 109894(endogenous control) 49 Probe 125322 (endogenous control) 50 Primer 147351 Primer 1504 52 Primer 1505 53 Primer 1506 54 Primer 1549 55 Primer1550 56 EPSP synthase from Agrobacterium sp. Cp4

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method for improving yield in a soybean plant comprising treatingsaid soybean plant with an effective amount of glyphosate, wherein saidsoybean plant comprises a 3560.4.3.5 event or a plant that is a progenyof a plant grown from seeds of ATCC Seed Deposit PTA-8287, wherein saidprogeny comprises the 3560.4.3.5 event, and wherein said soybean plantfurther comprises a polynucleotide sequence encoding a polypeptide thatimparts tolerance to glyphosate by a mode of action different thanglyphosate N-acetyltransferase.
 2. The method of claim 1, wherein saidpolynucleotide sequence encodes a glyphosate-tolerant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) polypeptide.
 3. Themethod of either claim 1, wherein the glyphosate is applied in a singletreatment or in successive treatments.
 4. The method of claim 1, whereinthe glyphosate is a glyphosate derivative comprising a salt or a mixtureof glyphosate salts selected from the group consisting ofmono-isopropylammonium glyphosate, ammonium glyphosate, and sodiumglyphosate.
 5. The method of claim 1, wherein the glyphosate orderivative thereof is used in a formulation comprising an adjuvantselected from the group consisting of: amines, ethoxylated alkyl amines,tallow amines, cocoamines, amine oxides, quaternary ammonium salts,ethoxylated quaternary ammonium salts, propoxylated quaternary ammoniumsalts, alkylpolyglycoside, alkylglycoside, glucose-esters,sucrose-esters, and ethoxylated polypropoxylated quaternary ammoniumsurfactants.
 6. The method of claim 1 wherein said polynucleotidesequence encodes a glyphosate oxidoreductase enzyme.
 7. The method ofclaim 1 wherein said polynucleotide sequence encodes a class II EPSPSenzyme.